A transgenic large mammal is produced by a method including the steps of obtaining one or more early embryos, selectively preparing an embryo having at least three cells, and preferably at a stage in development corresponding in time to the onset of transcription of the embryo""s paternal genome, and introducing isolated nucleic acid molecules into a blastomere of the selected embryo. The introduction of isolated nucleic acid molecules into such embryos results in the generation of transgenic large mammals at a significantly increased frequency as compared to introducing isolated nucleic acid molecules into zygotes or into the blastomeres of embryos at the one or two cell stage of development.

The first successful production of a transgenic mammal was accomplished using mice (Gordon et al., xe2x80x9cGenetic Transformation of Mouse Embryos by Microinjection of Purified DNAxe2x80x9d Proc. Nat;. Acad. Sci. USA, 77:7380-7384 (1980)). Transgenic large mammals (i.e., mammals of a species in which normal mature adults of either sex may attain a body mass of at least one kilogram) have also been produced, albeit with greater difficulty, and with a much lower frequency of transgenic offspring being obtained. The production of transgenic large mammals, including agriculturally valuable livestock, has thus been hindered by a low frequency of success (e.g., generally less than 3% in pigs) in obtaining transgenic offspring following introduction of exogenous nucleic acid molecules into zygotes, one celled embryos, or, in some cases, two celled embryos. This relatively low frequency increases the difficulty of obtaining any particular desired transgenic large mammal, with concomitant increases in time and expense.
The successful production of a variety of different transgenic large mammals has been reported. For example, transgenic large mammals have been made in which the animal produces an exogenous protein in milk, for example, tissue plasminogen factor expressed in goats and human anti hemophilic factor IX in sheep. (See, for review, Ebert, K. M. and J. P. Selgrath, xe2x80x9cChanges in Domestic Ungulates through Genetic Engineeringxe2x80x9d in Animal Applications in Mammalian Development, Cold Spring Harbor Laboratory Press, 1991. Transgenic Animal Technology: A Laboratory Handbook, Carl A. Pinkert, ed., Academic Press (1994). Transgenic swine have been produced which express porcine growth hormone under the control of promoter/enhancer elements originally isolated from Moloney murine leukemia virus or from cytomegalovirus (Ebert, K. M. et al., Animal Biotechnology 1:145-159 (1990).
Transgenic mammals are useful not only as improved agricultural stock or as a means of production for a protein, but are also valuable sources of altered cells, tissues, and organs, as well as valuable research tools, e.g., for discovering the mechanisms by which gene expression is controlled. (See, for example, Low, M. J. et al., Molecular Endocrinology 3:2028-2033 (1989), U.S. Pat. Nos. 5,573,940, 5,624,837, and 5,627,264, and PCT patent publications No. WO 95/04756, WO 95/23512, and WO 95/34202, which are incorporated herein by reference).
Transgenic mammals have been produced successfully by several methods, including introducing foreign DNA into the male pro-nucleus of a zygote (each pro-nucleus comprises only one haploid chromosomal complement); see, for example, Wagner et al., U.S. Pat. No. 4,873,191 (1989), disclosing transgenic mice). See also, Brinster, R. L. et al., Proc. Natl Acad. Sci. USA 82:4438-4442 (1985); Leder et al., U.S. Pat. No. 4,736,866 (1988)).
Transgenic large mammals have been obtained following injection of a transgene into both one and two celled embryos (Hammer, R E et al., Nature 315:680-683 (1985); Hammer, R E et al., J Anim Sci 63:269-278 (1986); Pursel et al., Proc Intl Cong Anim Reprod Artif Insem 11:480a-480c (1988); Pursel et al., J Reprod Fert Supl40:235-245(1990); Pursel et al., J Reprod Fertil Suppl 41:77-87 (1990); Mullins J J et al., Hypertension 22:630-633 (1993); Martin et al., xe2x80x9cProduction of Transgenic Swinexe2x80x9d pp 315-388 in Transgenic Animal Technology: A Laboratory Handbook, Carl A Pinkert, ed., Academic Press (1994); Seamark, R F Reprod Fertil Dev, 6:653-657 (1994); copending U.S. application Ser. No. 08/668,703, filed in the name of Karl M. Ebert on Jun. 24, 1996.
As a consequence of the low frequency with which transgenic large mammals have been obtained, new techniques that will allow a higher frequency of success in obtaining transgenic large mammals would be of great value, and are actively being sought.
The present invention provides methods of making transgenic large mammals that generate a higher frequency of transgenic offspring than has been heretofore achievable. These methods involve procedures in which at least one embryo is prepared and genetically modified. The genetic modification is accomplished by the introduction of at least one isolated nucleic acid molecule, and preferably at least one clone (i.e., a plurality of identical copies) of isolated nucleic acid molecules into an embryo.
Large Mammals: Preferred large mammals for use in the method of the invention are herbivores, and include ungulates (i.e., hoofed mammals such as pigs, cows, goats, sheep, horses, donkeys, deer, antelopes and the like) and more generally, livestock (i.e., mammals raised for agricultural purposes such as pigs, cows, goats, sheep, horses, rabbits and the like, and/or as beasts of burden such as donkeys, horses, elephants, camels, llamas, and the like). More preferable large mammals for use in the method of the invention are pigs, goats, sheep, and bovine cattle. Particularly preferred large mammals for use in the method of the invention are members of the genus Sus, with domestic pigs being the most highly preferred of these.
Embryos: In accordance with a preferred embodiment of the invention, embryos (or zygotes, which in accordance with certain aspects of the invention will be incubated in vitro and allowed to develop into embryos before genetic modification) are recovered from the reproductive tract of a donor female (e.g., a gilt or sow), typically from the oviduct or uterus of a mature, hormonally synchronized, ovulation induced female. The embryos are microscopically inspected to determine which are at the desired stage of development. Embryo development is typically measured and characterized in terms of the number of cells making up the embryo, which number is readily counted via microscopic inspection of a living embryo and increases throughout early embryonic development.
In accordance with the invention, embryos are prepared so as to provide at least one target embryo (i.e., an embryo into which at least one clone of isolated nucleic acid molecules is to be introduced). In accordance with the invention at least one of the at least one target embryos has at least three cells. Preferably the at least one target embryo that has at least three cells is obtained by preparing a group of embryos comprising the at least one target embryo that has at least three cells.
The genetic modification is accomplished by the introduction of at least one clone of isolated nucleic acid molecules into at least one blastomere of the at least one target embryo that has at least three cells. A blastomere is any of the cells of a multi-celled pre-gastrulation embryo, which cells each comprise a diploid nucleus. The at least one clone of isolated nucleic acid molecules preferably comprises at least one transgene (the at least one transgene-comprising clone being referred to hereinafter as a xe2x80x9ctransgene clonexe2x80x9d)
Preferably at least one clone of isolated nucleic acid molecules is introduced into a plurality of the blastomeres of each multi-cellular embryo in the group of embryos. More preferably the at least one clone of isolated nucleic acid molecules is introduced into the diploid nucleus of a blastomere of a target embryo that has at least three cells.
In accordance with certain of the preferred embodiments of the invention, at least one target embryo in the group of embryos is a target embryo comprising at least four cells. Preferably the target embryo comprising at least four cells is modified by the introduction of a transgene clone into at least one blastomere of the target embryo.
A preferred target embryo is a porcine embryo comprising at least three or at least four cells.
In accordance with certain of the preferred embodiments of the invention, at least one target embryo in the group of embryos is a target embryo comprising at least five or at least six cells. Preferably the target embryo comprising at least five or at least six cells is modified by the introduction of a transgene clone into at least one blastomere of the target embryo.
In accordance with certain of the preferred embodiments of the invention, at least one target embryo in the group of embryos is a target embryo comprising at least seven or at least eight cells. Preferably the target embryo comprising at least seven or at least eight cells is modified by the introduction of a transgene clone into at least one blastomere of the target embryo. A preferred target embryo comprising at least seven or at least eight cells is a bovine embryo.
In additional embodiments of the invention, at least one clone of isolated nucleic acid molecules is introduced into at least one embryo of a group of embryos and the criteria for target embryos are further limited so that the at least one clone of isolated nucleic acid molecules is not introduced into any embryos that do not contain at least two blastomeres. In certain embodiments of this subset of preferred embodiments, these criteria are additionally limited so that the at least one clone of isolated nucleic acid molecules is not introduced into any embryos that do not contain at least three blastomeres. In certain others of this subset of preferred embodiments, these criteria are also limited so that the at least one clone of isolated nucleic acid molecules is not introduced into any embryos that do not contain at least four blastomeres. In certain others of this subset of preferred embodiments, these criteria are also limited so that the at least one clone of isolated nucleic acid molecules is not introduced into any embryos that do not contain at least five or at least six blastomeres. In certain others of this subset of preferred embodiments, these criteria are additionally limited so that the at least one clone of isolated nucleic acid molecules is not introduced into any embryos that do not contain at least seven or at least eight blastomeres. In the embodiments where all of the target embryos comprise at least seven or at least eight blastomeres, a preferred target embryo is a bovine embryo.
Paternal Genome Activation: In accordance with a particularly preferred embodiment of the invention, the target embryo that has at least three cells is obtained and prepared in such a way that at least one clone of isolated nucleic acid molecules is introduced into one or more blastomeres having a paternal genome (the portion of a diploid embryo""s genome contributed by the sperm at fertilization) at a stage in the development of the embryo that is within one cell division of the point at which transcription of the paternal genome of the one or more blastomeres is initiated. This point in development may vary from species to species, but is believed to be generally uniform within a particular species of mammal.
Methods for determining the onset of transcription of the paternal genome in an embryo of any particular species of mammal are well known in the art. See, for example, Karp, G. et al. Dev Biol, 31:404-408 (1973); Geuskens et al. Cell. Different., 14:125-134 (1984); Davidson, E. H. Gene Activity In Early Development, 3rd edn. Academic Press, N.Y. (1986); Camous, S. et al. Biol Cell, 58:195-200 (1987); Tomanek, M. et al., et al. Anat Embryol, 180:309-316 (1989); and Telford, N. A. et al. Mol Reprod Dev, 26:90-100 (1990).
Embryo Culture: In certain of the preferred embodiments of the invention, embryos are harvested from a donor female and the at least one clone of isolated nucleic acid molecules is introduced into the blastomeres of only those embryos that have reached the desired stage of development (as indicated by the number of blastomeres making up the embryo), while those harvested embryos that have not yet reached the desired stage of development are maintained in culture until one or more cell divisions have occurred and at least one of the cultured embryos has reached the desired stage of development, after which point at least one clone of isolated nucleic acid molecules is introduced into at least one blastomere of the at least one cultured embryo.
Timing of Embryo Harvest: In accordance with certain preferred embodiments of the invention, embryos are harvested at a time after fertilization when at least one of the harvested embryos has a total number of blastomeres corresponding to the stage of development at which transcription of the paternal genome begins in that species. Preferably, embryos are harvested at a time after fertilization when the majority of the harvested embryos each has at least 2 blastomeres. In a particularly preferred embodiment, the majority of embryos each has a total number of blastomeres corresponding to a stage of development within one cell division of the point in development at which transcription of the paternal genome begins in that species. Additionally, the embryos may be characterized by being at a stage in development that is no earlier than one cell division before the onset of transcription of the paternal genome and is no later than one cell division after the first cell division following the onset of transcription of the paternal genome.
In general, this aspect of the invention involves embryo harvest later in gestation than is currently practiced in the art. Since gestation time in large mammals may change in response to a number of variables (e.g. seasonal changes), the optimum timing for embryo harvest in accordance with this aspect of the invention is best determined empirically, for example, as discussed in the following two paragraphs, where particulars of carrying out this aspect of the invention in pigs are discussed.
In pigs, as discussed above, the embryos to be genetically altered are preferably three- or four-cell embryos. Such three- or four-cell pig embryos can be collected by flushing the oviduct or the uterus of naturally inseminated (mated) or artificially inseminated gilts or sows 1.5 to 2.5 days after insemination, preferably about 48 hours after insemination, as described below in Example 1.
Typically, a cohort plurality of young sexually mature female large mammals (e.g., in pigs, gilts or sows, preferably giltsxe2x80x94non-pregnant nulliparous female pigs), are concurrently hormonally synchronized and preferably are then concurrently (typically one after another in prompt succession) inseminated. Preferably each female being prepared as an embryo donor is repeatedly inseminated, for example as described below in Example 1. By harvesting and microscopically inspecting embryos from a single female donor of the cohort so as to determine the number of cells per embryo, a skilled worker will readily be able to determine the stage of development of the harvested embryos and thereby determine when to harvest embryos from the remainder of the cohort so as to obtain the maximal yield of embryos at the desired stage of development.
In Vitro Development of Embryos: In accordance with certain preferred embodiments of the invention, a harvested embryo that has not yet reached the desired stage of development is incubated under appropriate culture conditions until it reaches the desired stage (at which point, in accordance with the invention, the embryo comprises at least three cells), at which time at least one clone of isolated nucleic acid molecules is introduced into one or preferably more than one of the blastomeres of the embryo. Culture conditions for embryos of large mammals have been described (see, for example, Berg et al., Zuchthyg, 24:184-189 (1989); Krisher et al., J Exp Zool, 249:235-239 (1989); Yoshida M, et al., Mol Reprod Dev 31:68-71 (1992); Hagen et al., J Anim Sci, 69:1147-1150 (1991); Crozet et al., J Reprod Fertil, 103:293-298 (1995)).
Development of the Transgenic Animal: After the at least one clone of isolated nucleic acid molecules is introduced into the at least one blastomere, the embryo is placed in an environment that will provide appropriate conditions for the development of the animal. Preferably the environment will allow for progression through embryonic and fetal development to term. Most preferably the environment is the reproductive tract of a female animal of the same species of animal as the embryo.
Introduction
A transgenic mammal is an mammal which has been engineered by the directed genetic alteration of the embryonic cells from which the mammal or one or more of its progenitors has developed. The directed genetic alteration is accomplished by the introduction into at least one of the embryonic cells (typically a cell of an early embryo, an embryonic stem cell, or a zygote) of one or more clones of isolated nucleic acid (preferably DNA) molecules. Preferably the one or more clones of isolated nucleic acid molecules comprise one or more transgenes.
As used herein, a transgene isxe2x80x941) a gene which is either synthesized in vitro or isolated from a biological source, and has been so prepared for subsequent introduction into a zygote or an embryonic cell, orxe2x80x942) the transgene (as per definition 1) or a biologically replicated copy thereof that is found within a cell of an animal subsequent to the introduction of the transgene (as per definition 1) into a zygote or an embryonic cell that participates in the development of the animal or at least one of its progenitors.
As used herein, a gene is an ordered sequence of nucleotides (or the like) that has a particular function, e.g., one that is transcribed and/or translated to form a specific functional product such as a protein or RNA molecule, or has a specific regulatory function in controlling the expression of other functional nucleic acid sequences.
Successful genetic alteration results in the integration of a transgene into the replicating complement of DNA of some or all of the cells of the animal (fetal and/or adult) that develops from the zygote or embryo. Preferably, the genetic alteration has detectable effects upon the cells containing the integrated transgene(s) and/or on the entire animal, e.g., over-expression of a protein or RNA molecule in the cells, expression of an exogenous protein or RNA molecule in the cells, alteration of the metabolism of the cells, and/or an alteration of the phenotype of the animal. Expression may occur for a limited period during ontogeny or throughout the life of the animal.
General Methods
The invention relates to methods of producing transgenic large mammals by introducing one or more clones of isolated nucleic acid molecules (preferably one or more transgene clones) into at least one cell of a multicellular pre-gastrulation embryo having at least three cells, preferably at a point in the development of the embryo that is within one cell division of the point in development at which transcription of the paternal genome begins.
A zygote is a fertilized egg in which the sperm nucleus (the haploid male pro-nucleus) and the egg nucleus (the haploid female pro-nucleus) have not yet fused to form a single diploid nucleus. The fusion of the haploid male and female pro-nuclei to form a diploid nucleus marks the transition from zygote to embryo. The onset of cleavage (a specialized form of cell division that occurs early in embryonic development) and the resulting multicellular stages of embryonic development cannot occur until the individually haploid zygote pro-nuclei fuse to form a diploid embryo nucleus.
Cells of the multicellular embryo are called blastomeres until the embryo has undergone gastrulation. An early blastomere is totipotent, meaning it has the capacity (even when isolated from the other blastomeres of the embryo) to develop into a complete, normal animal, given the appropriate conditions for development. Mammalian blastomeres are believed to be totipotent at least through the 8 cell stage of development (See review article, Papaioannou, V. E. and K. M. Ebert, In: Experimental Approaches to Mammalian Embryonic Development, Pedersen, R. and J. Rossant, eds., 1986.)
In accordance with certain of the preferred embodiments of the invention, embryo collection is timed so as to obtain a group of embryos the majority of which have reached at least the two- and preferably at least the three-cell stage of development.
In some large mammals, including for example pigs, it may be desirable to culture embryos (that have not yet reached the desired stage of development) and zygotes, and to monitor cell division in culture, so as to obtain embryos at the desired stage of development.
Any technique which allows for the addition of nucleic acid molecules into a blastomere can be utilized to accomplish the genetic modification of the target embryo so long as it is not destructive to the cell membrane, nuclear membrane or other essential structures of the blastomere, or to the blastomere in general. Such techniques include, but are not limited to, microinjection (e.g., into diploid nuclei), lipofection, gene gun techniques, and electroporation. Preferably, the isolated nucleic acid molecules are introduced into the diploid nucleus of the blastomere by microinjection.
The production of a transgenic mammal typically involves the use of a solution comprising a clone of isolated nucleic acid molecules, preferably comprising a transgene clone. The clone may be of biological origin (e.g., whole chromosomes, portions of chromosomes, chromosomal complexes, other forms of chromatin, plasmids, restriction fragments of plasmids, and the like) or may be synthetic or semi-synthetic nucleic acid molecules. In most cases the clone will be a transgene clone comprising a transgene transcription unit. Such a unit typically includes: 1) a promoter, 2) a transgene, and 3) a polyadenylation signal sequence. Other sequences, such as enhancer and intron sequences, can also be included if desired, e.g., to achieve increased gene expression.
The transgene transcription unit can be conveniently prepared, for example, by isolating a restriction fragment of a plasmid vector which expresses the protein of interest in mammalian cells. Preferably, the restriction fragment is free of sequences which direct replication in bacterial host cells (i.e., bacterial origin of replication sequences) since such sequences are believed to have deleterious effects on embryo viability. Nonetheless, a clone of an intact plasmid (a circular or linearized but unfragmented plasmid) although less preferred, may be used as the clone of isolated nucleic acid molecules to be introduced into an embryo blastomere in accordance with the invention. Thus, for example, genomic DNA or cDNA constructs may be injected in linear or supercoiled form.
With the exception of the lower limit imposed by the sizes of the fundamental building blocks from which they are made up (e.g., nucleotides, nucleosides, and the like), the size of the nucleic acid molecules to be introduced into a blastomere in accordance with the invention is not critical. This can therefore vary with the length of the nucleic acid sequence or sequences desired to be introduced. The usual size of a nucleic acid molecule to be introduced into a blastomere will be long enough to include at least one functional gene to be transferred, and may typically range from a few kb to on the order of 30 kb. Cosmids, YACs, and the like may be constructed so as to introduce nucleic acid molecules of up to hundreds of kb into a blastomere.
As an alternative to introducing one or more isolated clones of nucleic acid molecules that do not carry a functional eukaryotic origin of replication, and therefore must generally rely on integration into a chromosome or episome which has such an origin so as to be replicated during the cell division cycle, the transgene clone may be introduced as an artificial episomal chromosome (Sun, T-Q. et al., Nature Genetics 8:33-41 (1994)). Other types of natural or, preferably, artificial chromosomes may also be used as transgene clones in the methods of the invention if desired.
If a clone of isolated nucleic acid molecules is to be introduced into a blastomere by microinjection, it is preferably extensively purified first, for example, plasmids or restriction fragments may be purified by gel isolation followed by passage over an ion exchange resin such as an ELUTIP column (Schleicher and Schuell, Keene, N.H.), followed by dialysis against pyrogen free injection buffer (e.g., 10 mM Tris, pH7.4+0.1 mM EDTA in pyrogen free water). If the clone of isolated nucleic acid molecules is a chromosome or chromosome fragment clone, the purification method will preferably not separate the chromosomal proteins from the chromosomal nucleic acid molecules. In such cases, preferred purification methods include ultra-centrifugation (e.g., sucrose gradient centrifugation) and centrifugal elutriation.
The microinjection of zygote pro-nuclei or nuclei of one cell or, less commonly, two cell embryos to make transgenic mammals is widely practiced by those of skill in the art. As an example, transgenic swine are routinely produced by the microinjection of a transgene transcription unit into pig zygotes or embryos. See, for example, PCT Publication No. WO92/11757; Martin et al., xe2x80x9cProduction of Transgenic Swinexe2x80x9d pp 315-388 in Transgenic Animal Technology: A Laboratory Handbook, Carl A Pinkert, ed., Academic Press (1994).
Production of transgenic animals: At least one embryo (and preferably a plurality of embryos) is prepared. At least one (and preferably a majority) of the embryos has at least three cells. Preferably at least one (and more preferably a majority) of the embryos is at a stage of development within no more than one cell division from the stage at which transcription of the paternal genome begins.
The embryos are placed into a vessel (e.g., 1.5 ml microfuge tube) containing a small volume (e.g., approximately 0.5 ml) of a suitable embryo transfer medium (e.g., phosphate buffered saline with 10% fetal calf serum). Embryos of large mammals that have opaque embryos (e.g., pigs) are typically centrifuged to allow the visualization of the blastomere nuclei, e.g., for 4 minutes at 13,000xc3x97g in a microcentrifuge. Embryos are removed from the centrifuge tube (e.g., with a drawn and polished Pasteur pipette) and placed into a small dish (e.g., a 35 mm petri dish) for examination. If the cytoplasm is still opaque with lipid such that the nuclei are not sufficiently visible to allow visual verification of nuclear injection, the embryos may be centrifuged again for about 4 minutes.
Methods of microinjecting cells, such as zygotes and embryos, are well known in the art, and are described, for example, in Brinster, R. L. et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985). Such methods are also applicable to the microinjection of blastomere nuclei.
For microinjection, embryos can be placed on a surface, such as the center of the lid of a 100 mm petri dish, in culture medium. Typically a drop of medium (approximately 100 microliters) is used. Suitable media for the microinjection procedure in multicellular pre-gastrulation embryos are, for instance, modified BMOC-2 containing HEPES salts, as described in Ebert, K. M. et al. J. Embryol. Exp. Morph. 84:91-103 (1984); or PBS medium supplemented with 20% fetal calf serum (Jura, J. et al. Theriogenology 41:12S9-1266 (1994)) or Brinster""s medium plus 25 xcexcM HEPES buffer (pH 7.4) (Brinster, R. L. et al. Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985)). Of these, the first is most preferred. If desired, silicone oil or the like may be used to cover the drop of medium and to fill the lid to prevent the medium from evaporating.
An embryo to be injected is typically held in place by suction on a blunt holding pipet of a suitable diameter. Blastomere nuclei are then injected with a solution of isolated nucleic acid molecules via a sharp-tipped (1-2 xcexcm diameter, for example) injector pipet (injection needle). Swelling of the nucleus can generally be observed upon successful injection. The movements of the means for holding and injecting the cells are preferably controlled by appropriate micromanipulation instruments.
The process is best observed under a microscope (preferably an inverted microscope equipped with both a heated stage and enhanced contrast optics such as Hoffman modulation optics or Nomarski optics) using suitable magnification, e.g. 200-250xc3x97. Clones of isolated nucleic acid molecules can be injected into blastomere nuclei in a volume of 1-2 pl, optimally. More or less volume may be injected if desired, up to a maximum volume that does not cause irreparable damage to the nucleus or to the cell. Concentrations of the transgene clone may vary. Preferably the concentration will vary from approximately 1 to 4 ng/xcexcl for restriction fragments, with chromosomes and other larger molecules typically being at higher concentrations so as to adjust for the lower molarity of equivalent concentrations of such large molecules. Optimal concentrations of transgene clones to be used may be adjusted empirically, but higher integration frequencies have been observed in fertilized eggs when several hundred copies of a transgene are injected. (See, e.g., for heifers, McEvoy, T. G. et al., J. Reprod Fert Suppl, 43: 297-289 (1991)).
Embryos surviving the microinjection process, as judged, e.g., by microscopic observation of morphology, are preferably transferred to the reproductive tract of a recipient female shortly after introduction of the isolated nucleic acid molecules. In some instances it may be desirable for the surviving embryos to be cultured for a time and subsequently transferred. Surviving embryos are typically introduced into the oviduct or uterus, as appropriate so as to best match the developmental stage of the cultured embryo to the stage of pregnancy (or, although generally less preferred, pseudopregnancy) of the host female and the location of embryos in the reproductive tract of an animal at the corresponding stage of pregnancy). In some cases it may be more efficient to use donor females as recipients as well; i.e., subsequent to embryo collection, but while the animal is still undergoing the surgical procedure for embryo harvest, genetically altered embryos are introduced into the reproductive tract of the donor female, so as to utilize the donor as a recipeint. See, for example, Pursel et al., Thieriogenology, 46:201-209 (1996).
When required or desired, in vitro incubation for some period of time before transferring the embryo into an appropriate female host animal is preferably carried out with as little delay as possible. Preferably the delay is no more than 5 hours, more preferably the delay is no more than 4 hours, even more preferably the delay is no more than 3 hours, most preferably the delay is no more than 2 hours.
The host animal that serves as a surrogate mother can be any animal that can provide the appropriate hormonal and nutritional environment for the growth and development of the embryo to term. Such an animal can be a pregnant female with embryos at or close to the same stage of development as the surviving embryo or embryos to be transferred to her reproductive tract, or an animal at a stage in the estrous cycle in which the reproductive tract would be receptive to the introduction of an early embryo. For example, the estrous cycle of pigs can be hormonally synchronized using norgestomet implants, as described below in the examples.
Synchronization may also be achieved by other methods. For example, the use of ALTRENOGEST or the like in animal feed is well known in the art as a method for synchronizing large mammals. Typically, in pigs, ALTRENOGEST feeding is initiated 12-16 days following estrus and continued for 9 days, followed by the administration of Pregnant Mare""s Serum Gonadotropin (PMSG) one day after last ALTRENOGEST feeding. Human Chorionic Gonadotropin (hCG) is also administered as needed in conjunction with this regimen, for example as described below in Example 1 for the norgestimet implant method. Variations of these techniques allowing their application to the synchronization of a number of large mammal species are known in the art (see, for example, Selgrath et al., Theriogenology 34:1195-1205 (1990)xe2x80x94goats; Rexroad et al., Mol. Reprod. Dev. 1:164-169 (1989)xe2x80x94sheep), Hammer et al., Nature 315: 680-683 (1985)xe2x80x94(sheep, pigs, and rabbits) as are methods for the determination of optimal protocols for large animals for which such synchronization procedures have not been described in detail.
Alternatively, in some species (e.g., in rodents), the host female can be induced to a pseudopregnant state using appropriate treatments that are known in the art.
Preferably, the surrogate mother mammal is of the same species as the embryo. Usually, this is required for the development of the embryo to term. However, some exceptions are known. For example, one species of antelope can in some cases serve as surrogate mother for gestation of an embryo of a different species of antelope (Dresser, B. L. et al., Proc. Am. Assoc. Zool. Parks Aquar. 166-8 (1994)).
Applications and Further Considerations
Transgenic Animals: The invention can be used to modify a species (i.e., to create a new species variant). A new species variant can be obtained when a new genotype results from the introduction of the one or more clones of isolated nucleic acid molecules into one or more blastomeres of a target embryo and the new genotype occurs in the germline cells of an animal that develops from the target embryo so that the new genotype can be transmitted to the progeny of the animal.
The invention is particularly useful in the breeding of livestock of agricultural value, to obtain new species variants having genetic makeups that result in animals exhibiting more agriculturally and/or commercially desirable characteristics.
In some cases, it may be advantageous to use the transgenic large mammal embryo as a system to test for the timing and extent of expression of certain genes during the development of the animal. This can be done using known methods in the art. One method is, for example, recovering an embryonic or fetal animal, isolating polyA+ RNA from one or more tissue types of interest, and testing the polyA+ RNA (e.g., using hybridization with a labeled nucleic acid probe under maximally stringent conditions) for the presence of sequences homologous to the transgene.
Nucleic Acid Molecules: The one or more clones of isolated nucleic acid molecules may be obtained from numerous biological sources, such as animals or plants, viruses, bacteria, or protozoa; or may be of synthetic origin. The one or more clones may be synthetic equivalents of naturally occurring genetic material or may be totally new synthetically produced genetic material. The one or more clones of isolated nucleic acid molecules can be from the same species as the blastomere into which it is to be introduced, or from a different species.
Depending upon the particular trait or traits which are desired in the animal, it will generally be necessary to include in the introduced nucleic acid molecules the controlling elements (e.g., promoters, enhancers, polyadenylation sites, transcription termination sites, ribosome binding sites, and the like) responsible for expression of the gene(s) coding for the gene product that will affect the trait.
In some cases, it may be desirable to introduce back into the same species, in unmutated form, a gene originally isolated from that species. More typically, if it is desired to introduce back into the same species a gene originally isolated from that species, the gene will have been altered (mutated) to produce a different amount of gene product and/or an altered gene product compared to that produced in normal (e.g., non-transgenic) animals. In many cases it may be desirable to introduce back into the same species a gene originally isolated from that species which has been put under the control of controlling elements, either synthetic or isolated from a different organism and/or a different gene. In some instances it may be desirable to include a control region that will activate the gene when the transgenic animal is exposed to a stimulus other than one of the natural stimuli that activate or deactivate the gene.
Techniques for obtaining segments of DNA by gene excision, splicing, synthesis, isolation, purification, cloning and the like are well known in the art. Enzymes used in such processes, vectors and hosts for cloning of recombinant nucleic acid molecules, screening and selection of desired nucleic acid molecules and detection and analysis of expression of cloned genes are also well known in the art. (See, for example, Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley and Sons Inc., (1997)).
The number of nucleic acid molecules which constitute the one or more clones of isolated nucleic acid molecules that is introduced into a blastomere will depend upon several factors, including (for transgene clones) the number of copies of the transgene per nucleic acid molecule. The total amount of isolated nucleic acid molecules introduced is preferably an amount which enables the integration of an isolated nucleic acid molecule into the genome of the blastomere. In cases where the one or more clones of isolated nucleic acid molecules comprise a transgene clone, total amount of isolated nucleic acid molecules introduced is preferably an amount which allows incorporation of at least one transgene of the transgene clone to occur in such a way that the at least one transgene can be expressed. Theoretically, only one copy of a particular transgene is required. However, it is preferable that numerous copies of the nucleic acid molecules which constitute transgene clone are introduced, for example, 100-1,000 copies, in order to insure that at least one copy of at least one transgene becomes integrated in a functional state.
For example, a successful result of prior art methods of injecting the male pronucleus of a zygote of a mouse with multiple copies of linear DNA nucleic acid molecules which constitute a transgene clone has been, typically, a transgenic mouse with all or some of its cells containing a tandem array of copies of the injected nucleic acid molecules (and the transgene therewithin) integrated at a single random site in one of its chromosomes (Palmiter, R. D. and R. L. Brinster Ann. Rev. Genet. 20:4 65-499 (1986)).
Generally, the chief advantage to having more than one functioning copy of each of the introduced transgenes is enhanced expression. There are also instances where more than one functional copy of the transgene clone may be undesirable, for example when a transgene works in conjunction with endogenous nucleic acid sequences of the organism to produce a particular product.
Identifying Transgenic Offspring: Cell or tissue samples may be taken from the animal developing from the embryo into which the one or more clones of isolated nucleic acid molecules has been introduced to test for the presence of the introduced nucleic acid molecules. Depending on the type of large mammal, cells obtained from the embryo, fetus, neonate, developing offspring or adult can be tested for the presence of the introduced nucleic acid molecules by well-known techniques of molecular biology. Such techniques involve, for example, obtaining a sample of DNA from cells (typically and preferably somatic cells) obtained from the fetus, neonate or developing offspring (e.g., from a segment of tail or a snippet of ear) and testing it for the presence of the introduced nucleic acid molecules. Such testing may be accomplished, for example, by nucleic acid hybridization using a specific probe, or by using oligonucleotides (e.g., as primers) for the specific amplification of the transgene, e.g., by PCR (or another amplification method), or by other suitable methods. Such additional methods may include the identification of a phenotypic effect of the introduced nucleic acid molecules. This may be identified either by detection of the appearance of a previously absent gene product or by detection of altered levels of a native gene product. Such detection may be accomplished, for example, by probing with a gene product specific antibody.
Phenotypic alterations may also be detected as alterations in the expression of other genes interacting with the one or more isolated nucleic acid molecules that have been used to produce the transgenic animal. For example, the addition of one or more transgenes in the form of a new promoter and/or controlling element, or a fragment of a promoter and/or controlling element introduced to supplement an existing promoter and/or controlling element, may effect a detectable phenotypic alteration.
Identifying Germ Line Transmission: Transgenic animals capable of germ-line transmission are particularly desirable, as they allow the creation of and perpetuation of new species variants. Such transgenic animals can be tested for by various means well known in the art, for example through the analyses of the DNA of germ cells (e.g., as described above) and/or by breeding experiments.
Additional Limitations: The physical effects of the alteration in the genome of the altered blastomere must not be so great as to destroy the viability of the blastomere. The biological limit of the number and variety of isolated nucleic acid molecules introduced will vary depending upon the particular blastomere and the functions of any transgenes incorporated in the isolated nucleic acid molecules. The genetic material of the resulting blastomere must be biologically capable of initiating and maintaining the differentiation and development of the blastomere. When the one or more clones of isolated nucleic acid molecules are in the form of chromatin, chromatinosomes, or chromosome fragments, the addition of no more than one chromosome (and, more typically, less than one chromosome, i.e., part of a chromosome) is generally preferred in order to limit the potentially deleterious effects of extra chromosomes upon cell division and viability.

Methods for producing transgenic pigs by microinjecting a blastomere


A communication apparatus which communicates data with plural other communication apparatuses by performing wireless communication includes: plural antennas; a communication level obtainment unit which obtains communication levels for the antennas obtained when the communication apparatus performed the wireless communication with the other communication apparatuses; a count unit which counts, for each of the antennas, the number of established communications that is the number of one or more of the other communication apparatuses which successfully established communication through the antenna; an antenna selection unit which selects, when the total number of antennas whose numbers of established communications are greatest is more than one, one of the antennas corresponding to a communication level that is highest among communication levels of plural antennas of the other communication apparatus corresponding to the lowest communication level; and a transmission unit which broadcasts predetermined data to the other communication apparatuses through the selected antenna.

In recent years, besides conventional wire communication, attention has been focused on wireless communication that is more convenient. In wireless communication, a technique (diversity technique) is available in which plural antennas are provided and used in communication for the purpose of expanding a communication area or suppressing an effect of an interference wave.
In such a network including (i) an access point having plural antennas and (ii) plural stations, when broadcasting data to the stations, the access point is required to select, among the antennas, an antenna for use in broadcasting data to the stations. The conventional method for selecting an antenna for broadcasting includes a method of detecting, for each of the antennas of the access point, the number of errors in data (signals) received by the access point through the antenna, and selecting the antenna having less errors from among the antennas (see Patent Literature (PTL) 1).

Communication apparatus and communication method

PANASONIC INTELLECTUAL PROPERTY MANAGEMENT CO., LTD.


A method and apparatus are disclosed for generating random numbers using the meta-stable behavior of flip-flops. A flip-flop is clocked with an input that deliberately violates the setup or hold times (or both) of the flip-flop to ensure meta-stable behavior. When a meta-stable event is detected, an output bit is provided as a random bit. An even random number distribution is obtained by “marking” half of the zeroes input to the flip-flop as “ones” and the other half of the zeroes as “zeroes.” In addition, half of the ones are marked as “ones” and the other half of the ones are marked as “zeroes.” The marking signal is uncorrelated to any noise to a high probability using a linear feedback shift register.

Flip-flops and latches are widely used in computers and other electronic devices, for example, as sampling, counting and storage elements. A number of flip-flop types have been developed, such as D-type flip-flops ("data"), R-S latches ("reset and set"), J-K flip-flops (having J and K inputs) and T flip-flops (having only one input). A D-type flip-flop, for example, is a clocked flip-flop whose output is delayed by one clock pulse.
A conventional R-S latch 100 is shown in FIG. 1A. As in FIG. 1A, the R-S latch 100 is comprised of two NOR gates 110 and 120. The outputs of the two NOR gates 110, 120 are cross-connected to a respective input of the opposite NOR gate. Thus, NOR gate 110 receives the output of NOR gate 120 and a reset 30 signal, R, as inputs. Likewise, NOR gate 120 receives the output of NOR gate 110 and the set signal, S, as inputs.
More recently, the simple latches shown in FIG. 1A have been replaced by edge-triggered flip-flops, such as the D-type flip-flop 150 is shown in FIG. 1B. Such D-type flip-flops are often used to detect the logic state of an asynchronous digital signal having an unpredictable timing relative to the clock signal. A rising signal is applied to the clock input, CLK, of the flip-flop 150, while a digital logic level of the asynchronous signal to be detected is directed to the D input. The detected signal is then produced on the Q output line. As long as the clock does not rise again, the output Q does not change. Thereafter, the flip-flop 150 simply changes state to the value on the D input whenever the CLK signal rises (so long as the reset signal is tied permanently to ground).
The latches 100 shown in FIG. 1A are susceptible to meta-stability. For a detailed discussion of meta-stability, see, for example, Application Note, A Meta-Stability Primer, AN219, Philips Semiconductors (Nov. 15, 1989), incorporated by reference herein. Generally, meta-stability can occur when both inputs to a latch 100 are set at a high logic value ("11"), and are then reset to a low logic value ("00") Under these conditions, the latch outputs can oscillate unpredictably in a statistically known manner. In theory, the latch 100 can oscillate indefinitely. In practice, however, the latch 100 will randomly shift and arrive at a random output value of either logic low or high. Typically, these meta-stable values are subsequently detected by other circuitry in a given application and can be interpreted as different logic level states or assume an intermediate state that can be misinterpreted by other logic gates.
In addition, the edge-triggered flip-flop 150 shown in FIG. 1B can become meta-stable when the setup or hold times of the flip-flop are violated. Edge-triggered flip-flops 150 are susceptible to meta-stability because inside every edge-triggered flip-flop 150 there is a latch 100 being fed by the edge detection circuitry. If the setup or hold times are violated then the internal latch 100 will observe inputs that can trigger the meta-stable state.
Many applications and electronic devices require random numbers, including games of chance, such as poker, roulette, and slot machines. In particular, numerous cryptographic algorithms and protocols depend on a non-predictable source of random numbers to implement secure electronic communications and the like. A random number generator should generate every possible permutation in the designated range of numbers. In addition, the random number generator should not be biased and should generate any given number with the same probability as any other number. Moreover, the random number generator should generate random numbers that cannot be predicted, irrespective of the size of the collection of previous results. Thus, the random numbers should be completely unpredictable and non-susceptible to outside influences.
U.S. patent application Ser. No. 09/519,549, filed Mar. 6, 2000, entitled "Method and Apparatus for Generating Random Numbers Using Flip-Flop Meta-Stability," discloses a method and apparatus for generating random numbers using the meta-stable behavior of flip-flops. A flip-flop is clocked with an input that deliberately violates the setup or hold times (or both) of the flip-flop to ensure meta-stable behavior. A bit is collected whenever there is an error. If meta-stability occurs more frequently with one binary value (either zero or one) for a given class of flip-flops, an even random number distribution is obtained by "marking" half of the zeroes as "ones" and the other half of the zeroes as "zeroes." In addition, half of the ones are marked as "ones" and the other half are marked as "zeroes".
Marking input bits in this manner theoretically provides an even distribution of random output bits. While meta-stability occurs on a random basis, it has been found that the duration and occurrence of meta-stability is affected by noise. Thus, if the noise is correlated to the marking signal, then the output of the random number generator will not be random. A need therefore exists for a method and apparatus for generating random numbers using meta-stability that is not influenced by noise or other outside forces. A further need exists for a method and apparatus for generating random numbers using meta-stability that that uses a marking signal that is uncorrelated with a high probability to any noise in the system.

Method and apparatus for decorrelating a random number generator using a pseudo-random sequence


A system for probing a DUT is disclosed, the system having a pulsed laser source, a CW laser source, beam optics designed to point a reference beam and a probing beam at the same location on the DUT, optical detectors for detecting the reflected reference and probing beams, and a collection electronics. The beam optics is a common-path polarization differential probing (PDP) optics. The common-path PDP optics divides the incident laser beam into two beams of orthogonal polarization—one beam simulating a reference beam while the other simulating a probing beam. Both reference and probing beams are pointed to the same location on the DUT. Due to the intrinsic asymmetry of a CMOS transistor, the interaction of the reference and probing beams with the DUT result in different phase modulation in each beam. This difference can be investigated to study the response of the DUT to the stimulus signal.

1. Field of the Invention
The present invention relates to an apparatus and method for probing integrated circuits using laser illumination.
2. Description of the Related Art
Probing systems have been used in the art for testing and debugging integrated circuit (IC) designs and layouts. Various laser-based systems for probing IC's are known in the prior art. While some description of the prior art is provided herein, the reader is encouraged to also review U.S. Pat. Nos. 5,208,648, 5,220,403 and 5,940,545, which are incorporated herein by reference in their entirety. Additional related information can be found in Yee, W. M., et al. Laser Voltage Probe (LVP): A Novel Optical Probing Technology for Flip-Chip Packaged Microprocessors, in International Symposium for Testing and Failure Analysis (ISTFA), 2000, p 3-8; Bruce, M. et al. Waveform Acquisition from the Backside of Silicon Using Electro-Optic Probing, in International Symposium for Testing and Failure Analysis (ISTFA), 1999, p 19-25; Kolachina, S. et al. Optical Waveform Probing—Strategies for Non-Flipchip Devices and Other Applications, in International Symposium for Testing and Failure Analysis (ISTFA), 2001, p 51-57; Soref, R. A. and B. R. Bennett, Electrooptical Effects in Silicon. IEEE Journal of Quantum Electronics, 1987. QE-23(1): p. 123-9; Kasapi, S., et al., Laser Beam Backside Probing of CMOS Integrated Circuits. Microelectronics Reliability, 1999. 39: p. 957; Wilsher, K., et al. Integrated Circuit Waveform Probing Using Optical Phase Shift Detection, in International Symposium for Testing and Failure Analysis (ISTFA), 2000, p 479-85; Heinrich, H. K., Picosecond Noninvasive Optical Detection of Internal Electrical Signals in Flip-Chip-Mounted Silicon Integrated Circuits. IBM Journal of Research and Development, 1990. 34(2/3): p. 162-72; Heinrich, H. K., D. M. Bloom, and B. R. Hemenway, Noninvasive sheet charge density probe for integrated silicon devices. Applied Physics Letters, 1986. 48(16): p. 1066-1068; Heinrich, H. K., D. M. Bloom, and B. R. Hemenway, Erratum to Noninvasive sheet charge density probe for integrated silicon devices. Applied Physics Letters, 1986. 48(26): p. 1811.; Heinrich, H. K., et al., Measurement of real-time digital signals in a silicon bipolar junction transistor using a noninvasive optical probe. IEEE Electron Device Letters, 1986. 22(12): p. 650-652; Hemenway, B. R., et al., Optical detection of charge modulation in silicon integrated circuits using a multimode laser-diode probe. IEEE Electron Device Letters, 1987. 8(8): p. 344-346; A. Black, C. Courville, G Schultheis, H. Heinrich, Optical Sampling of GHz Charge Density Modulation in SIlicon Bipolar Junction Transistors Electronics Letters, 1987, Vol. 23, No. 15, p. 783-784, which are incorporated herein by reference in their entirety.
As is known, during debug and testing of an IC, a commercially available Automated Testing Equipment, also known as an Automated Testing and Evaluation (ATE) tester, is used to generate test patterns (also referred to as test vectors) to be applied to the IC device under test (DUT). When a laser-based system is used for probing, the DUT is illuminated by the laser and the light reflected from the DUT is collected by the probing system. As the laser beam strikes the DUT, the laser beam is modulated by the response of various elements of the DUT to the test vectors. This has been ascribed to the electrical modulation of the free carrier density, and the resultant perturbation of the index of refraction, of the material. Accordingly, analysis of the reflected light provides information about the operation of various devices in the DUT.
FIG. 1 is a general schematic depicting major components of a laser-based voltage probe system architecture, 100, according to the prior art. In FIG. 1, dashed arrows represent optical path, while solid arrows represent electronic signal path. The optical paths represented by curved lines are generally made using fiber optic cables. Probe system 100 comprises a dual laser source, DLS 110, an optical bench 112, and data acquisition and analysis apparatus 114. The optical bench 112 includes provisions for mounting the DUT 160. A conventional ATE tester 140 provides stimulus signals and receives response signals 142 to/from the DUT 160 and provides trigger and clock signals, 144, to the time-base board 155. The time-base board 155 synchronizes the signal acquisition with the DUT stimulus and the laser pulses. Workstation 170 controls as well as receives, processes, and displays data from the signal acquisition board 150, time-base board 155, and the optical bench 112.
The various elements of probe system 100 will now be described in more detail. Since temporal resolution is of high importance in testing DUT's, the embodiment of FIG. 1 utilizes prior art pulsed lasers, wherein the laser pulse width determines the temporal resolution of the system. Dual laser source 110 consists of two lasers: a pulsed mode-locked laser, MLL 104, source that is used to generate 10-35 ps wide pulses, and a continuous-wave laser source, CWL 106, that can be externally gated to generate approximately 1 μs wide pulses. The MLL 104 source runs at a fixed frequency, typically 100 MHz, and must be synchronized with the stimulus 142 provided to the DUT 160, via a phase-locked loop (PLL) on the time-base board 155, and the trigger and clock signals 144 provided by the ATE tester. The output of the DLS 110 is transmitted to the optical bench 112 using fiber optics cable 115. The light beam is then manipulated by beam optics 125, which directs the light beam to illuminate selected parts of the DUT 160. The beam optics 125 consists of a Laser Scanning Microscope (LSM 130) and beam manipulation optics (BMO 135). The specific elements that are conventional to such an optics setup, such as objective lens, etc., are not shown. Generally, BMO 135 consists of optical elements necessary to manipulate the beam to the required shape, focus, polarization, etc., while the LSM 130 consists of elements necessary for scanning the beam over a specified area of the DUT. In addition to scanning the beam, the LSM 130 has vector-pointing mode to direct the laser beams to anywhere within the field-of-view of the LSM and Objective Lens. The X-Y-Z stage 120 moves the beam optics 125 relative to the stationary DUT 160. Using the stage 120 and the vector-pointing mode of the LSM 130, any point of interest on the DUT 160 may be illuminated and probed.
For probing the DUT 160, the ATE 140 sends stimulus signals 142 to the DUT, in synchronization with the trigger and clock signals provided to the phase-locked loop on the time-base board 155. The phase-lock loop controls the MLL 104 to synchronize its output pulses to the stimulus signals 142 to the DUT. MLL 104 emits laser pulses that illuminate a particular device of interest on the DUT that is being stimulated. The reflected light from the DUT is collected by the beam optics 125, and is transmitted to photodetector 138 via fiber optic cable 134. The reflected beam changes character depending on the reaction of the device to the stimulus signal. To monitor incident laser power, for purposes of compensating for laser power fluctuations, for example, optical bench 112 provides means to divert a portion of MLL 104 incident pulse to photodetector 136 via fiber optic cable 13. The output signal of the photodetectors 132, 134 is sent to signal acquisition board 150, which, in turn, sends the signal to the controller 170. By manipulation of the phase lock loop on the time-base board 155, controller 170 controls the precise time position of MLL 104 pulses with respect to DUT 160 stimulus signals 142. By changing this time position and monitoring the photodetectors signals, the controller 170 can analyze the temporal response of the DUT to the stimulus signals 142. The temporal resolution of the analysis is dependent upon the width of the MLL 104 pulse.
A major difficulty encountered by all laser-base probe systems is deciphering the weak modulation in the reflected signal, which is caused by the response of the DUT to the stimulus. Another difficulty is noise introduced into the signal by the DUT's vibrations. Various beam manipulation optic, 135, designs have been used in the art in an attempt to solve these difficulties. FIG. 2 is a diagram illustrating standard amplitude detection mode used in the prior art. In FIG. 2, a laser probe is used to probe specific device 210, such as a transistor's gate or drain, in a DUT. A beam splitter 220 is used to separate the reflected beam from the incident laser beam. Amplitude modulation due to DUT interaction with the laser beam can be detected directly using a photodetector. However, DUT vibrations cause amplitude variations that are much stronger than the variation from the DUT activity of interest. This necessitates noise rejection schemes to make such an arrangement practical. One scheme implemented in the prior art is dual-laser noise rejection. In dual-laser noise rejection, the pulses from CWL 106 are used to measure the DUT 160 vibrations. The CWL measurements are then used to normalize the MLL measurements of the DUT activity.
The DUT interaction with the laser beam may cause changes mostly in the phase of the reflected laser beam, not its amplitude. Consequently, the signal strength may be too weak for pure amplitude detection. Various phase detection schemes have been developed for the beam manipulation optics 135. FIG. 3 is a diagram illustrating a phase detection scheme using a Michelson Interferometer arrangement to convert phase to amplitude. This scheme is also referred to as Phase-Interferometric Detection, or PID, mode. To detect phase modulations, a portion of the incident beam from the laser source is directed into a reference arm consisting of a lens 340 and a mirror 330, using beam splitter 320. The remaining portion of the incident beam is directed to a specific device of interest on the DUT, and upon reflection it is modulated according to the DUT's response to a stimulus signal. The light beam, 355, reflected by the DUT, and the light beam, 335, reflected by the reference arm mirror 330, are spatially recombined into a single beam 365 so that they can interfere (light beams 355 and 335 are shown spatially separated in FIG. 3 for illustrative purposes). The interference effect converts relative phase differences between the reflected beam 355 and the reference arm beam 335 into amplitude differences in resultant beam 365, which can then be detected by a photodetector.
While this arrangement helps detect phase variations caused by the DUT, using this optical arrangement exposes the system to additional noise source from phase variations caused by DUT vibrations. The DUT vibrations still modulate reflected DUT beam amplitude, but now also modulate the DUT beam phase, which generates larger resultant beam 365 amplitude modulations. To compensate for this phase noise, a modified dual-laser noise rejection scheme is used. In this modified scheme, the CWL resultant signal is used in a feedback loop to control reference arm mirror 330 position. By striving to maintain constant CWL resultant signal, the feedback loop drives reference arm mirror 330 to track DUT vibrations in order to maintain a constant quiescent phase offset value between DUT and reference arms. Fine control of mirror 330 position for the feedback loop is provided via a piezo electric transducer element (not shown). Additional adjustments (not shown) that are required in order to get best performance include reference arm power control and coarse reference arm mirror position control. Reference arm power control allows the reflected power from the DUT and reference arm mirror to be matched. Coarse reference arm mirror position control allows DUT and reference arm optical path lengths to be nominally equalized, a necessity for the operation of the modified dual-laser noise reduction scheme due to wavelength differences between MLL and CWL used in the prior art. Means to align optics to ensure overlap of reflected DUT and reference arm beams are also necessary for best performance.
FIG. 4 is a diagram illustrating another scheme, generally known as (spatial) differential probing (SDP) for phase detection. A Wollaston prism, contained within beam manipulation optics 430, is used to generate the two spatially separated beams, 422, 424. The two beams, 422 and 424, have orthogonal, linear polarization states (denoted by the dots and arrows of beams 424 and 422, respectively). One beam, e.g., 422, is directed to the DUT active device of interest; while the other beam, say, 424, can be directed to either an inactive device or region, or to an active device with complementary modulation. The advantage of the latter option is that the measured signal modulation is increased because the relative phase modulation between the two beams is doubled. In the particular example of FIG. 4, the two beams are directed to the drains of the p- and n- FETs of an inverter, which generates complementary modulations of the beam. Beam manipulation optics 430 spatially recombines the two reflected beams, 435, 455, and converts them to the same polarization state so that they may interfere with each other to generate amplitude modulated resultant beam 465. Beam manipulation optics 430 also provides means to introduce a phase offset between the two beams so that the interference condition can be optimized for maximum signal sensitivity. Using this scheme the phase noise is reduced relative to the scheme illustrated in FIG. 3, because each beam is directed at the DUT, so that the DUT vibrations will tend to modulate the phase of both beams similarly.
As can be understood, various IC's have different layouts, and different devices within an IC's have different dimensions and surroundings. Therefore, using this embodiment, for each device to be probed the user needs to decide where to place each beam within the chip. Moreover, since the beam needs to be placed at various locations in the chip, the system needs to be designed so that the beam separation is adjustable, which complicates the optics design. Additionally, the intensity ratio of the beams must be variable since the reflectivity of the regions where they are placed can differ. Power matching between the two beams is required for best results.
Experience with devices as depicted in FIG. 4 has shown that DUT vibrations can still generate amplitude fluctuations if the two laser beams are not incident on identical structures. Due to variations in the layout and dimensions of the DUT, it is difficult to find identical structures for probing within the range of adjustment of the two laser beams. Differences in structure cause differences in reflection as the DUT is vibrated. To reduce the effects of these vibrations, the dual-laser noise reduction scheme can be used. However, the different polarization states of the two beams also makes them respond differently to time-varying birefringence in the DUT or in the optics of the system. Time varying birefringence in the DUT can be caused by stress variations due to mechanical vibrations, for example.
FIG. 5 is a diagram illustrating time differential probing (TDP) scheme for phase detection. Two pulsed beams, 522 and 524, are generated by splitting a single pulse beam in two and time shifting one relative to the other by a small amount (approx. 10-100 ps) before DUT interaction. Time shifting can be achieved by passing one beam through an optical delay line (such as a thick piece of glass) contained within beam manipulation optics, 526. The two beams are spatially recombined after splitting so that they traverse a common path to the DUT. After DUT reflection, the opposite beam is passed through the optical delay line to remove the relative delay between the two beams. The two beams are then allowed to interfere, 530. Since the two beams traverse a common path, DUT vibrations largely modulate both identically, making this scheme inherently vibration insensitive. However, the optics required to generate the delay, and then reverse its effect for the reflected beams, are relatively complicated. Additionally, the beams are not completely identical. They have orthogonal polarization states so DUT interactions are not truly identical (birefringence effects can cause non-common mode variations of the beams). In this scheme, the resulting 'waveforms' are derivatives of the signal at the probed device in the DUT and typically consist of positive and negative going peaks in the case of probed logic signals, for example. When both beams are position in time on logic highs or lows, the resultant beam intensity is the same since both beams are phase shifted identically in each case. Only when the time shifted beams span a logic transition (one beam on a logic high, the other beam on a logic low) or part of a logic transition, does a phase difference result. This arrangement requires high temporal resolution of the sampling to ensure that each logic transition of interest is spanned by the time separated beams. This can limit the maximum time span of a sampling window that can be used, or may require additional acquisition time to fill the desired sampling window with enough density of sampling points. In addition, the temporal resolution (or measurement bandwidth) of this scheme is limited by the pulse separation used instead of by the width of the laser pulses. Since larger pulse separations typically give better signal strength, this scheme suffers from a measurement bandwidth versus signal strength trade-off.
Accordingly, there is a need in the art for a system that will allow improved laser probing of a DUT, while simplifying operation and minimizing the system's complexity and cost.

This patent is subject to a terminal disclaimer.

Apparatus and method for probing integrated circuits using polarization difference probing


Direct patterning of nanometer scale features by interferometric lithography using a 46.9 nm laser is described. Multiple exposures using a Lloyd's mirror interferometer permitted printing of arrays having 60 nm FWHM features.

Regular arrays of metallic or semiconductor nanoscale pillars and rings having sizes below 100 nm have recently attracted attention due to their unique optical, electrical and magnetic properties. Different strategies have been used for the fabrication of such arrays, including photon and electric beam lithography, self assembly of nanospheres or replication by embossing, molding or printing with master stamps. In the case of self assembly, the arrangement of the nanoscale structures is often random, or organized in reduced areas. The replication using master stamps has demonstrated feature sizes below 100 nm and large area coverage. However, a different master is required for each pattern, thereby restricting the versatility of the method.
An approach commonly used to print nanoscale periodic features is interferometric lithography (IL). This maskless photolithography technique is based on the activation of a photoresist by the interference pattern generated by two or more spatially coherent light beams. IL can efficiently print periodic patterns in a sensitive photoresist coated substrate with resolution approaching half of the wavelength of the illumination. In this approach, reducing the wavelength of the illumination provides a direct and simple path to realizing interference patterns having dimensions of tens of nanometers and below. This has been the principal motivation for using ultraviolet, shorter wavelength extreme ultraviolet light (EUV) or soft x-ray light (SXR) for such applications. The versatility of EUV/SXR IL for patterning different patterns and realizing small features has been demonstrated using 13 nm wavelength light from a synchrotron source. However, the widespread use of this technique in applications requires its implementation on a more compact and readily accessible apparatus.
Accordingly, it is an object of the present invention to provide an apparatus and method for patterning nanoscale features in materials by lithography using extreme ultraviolet/soft x-ray (EUV/SXR) interferometry.
Additional objects, advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

Nanometer-scale lithography using extreme ultraviolet/soft x-ray laser interferometry

Colorado State University Research Foundation


A method for decoding a data stream containing audio/video substreams and control substreams comprises buffering nodes having the possibility to buffer multiple data packets in the same buffer. This may be achieved by having separate parameters for the allocated buffer size and any stored packet. Thus, not only multiple packets may be stored in the buffering node, but also such node may exist while its buffer is empty, so that the node may be reused later. This is particularly useful for buffering and selectively accessing multiple audio packets in MPEG-4 audio nodes or sound nodes.

In the MPEG-4 standard ISO/IEC 14496, in particular in part 1 Systems, an audio/video (AV) scene can be composed from several audio, video and synthetic 2D/3D objects that can be coded with different MPEG-4 format coding types and can be transmitted as binary compressed data in a multiplexed bitstream comprising multiple substreams. A substream is also referred to as Elementary Stream (ES), and can be accessed through a descriptor. ES can contain AV data, or can be so-called Object Description (OD) streams, which contain configuration information necessary for decoding the AV substreams. The process of synthesizing a single scene from the component objects is called composition, and means mixing multiple individual AV objects, e.g. a presentation of a video with related audio and text, after reconstruction of packets and separate decoding of their respective ES. The composition of a scene is described in a dedicated ES called 'Scene Description Stream', which contains a scene description consisting of an encoded tree of nodes called Binary Information For Scenes (BIFS). 'Node' means a processing step or unit used in the MPEG-4 standard, e.g. an interface that buffers data or carries out time synchronization between a decoder and subsequent processing units. Nodes can have attributes, referred to as fields, and other information attached. A leaf node in the BIFS tree corresponds to elementary AV data by pointing to an OD within the OD stream, which in turn contains an ES descriptor pointing to AV data in an ES. Intermediate nodes, or scene description nodes, group this material to form AV objects, and perform e.g. grouping and transformation on such AV objects. In a receiver the configuration substreams are extracted and used to set up the required AV decoders. The AV substreams are decoded separately to objects, and the received composition instructions are used to prepare a single presentation from the decoded AV objects. This final presentation, or scene is then played back.
According to the MPEG-4 standard, audio content can only be stored in the 'audioBuffer' node or in the 'mediaBuffer' node. Both nodes are able to store a single data block at a time. When storing another data block, the previously stored data block is overwritten.
The 'audioBuffer' node can only be loaded with data from the audio substream when the node is created, or when the 'length' field is changed. This means that the audio buffer can only be loaded with one continuous block of audio data. The allocated memory matches the specified amount of data. Further, it may happen that the timing of loading data samples is not exactly due to the timing model of the BIFS decoder.
For loading more than one audio sample, it is possible to build up an MPEG-4 scene using multiple 'audioBuffer' nodes. But it is difficult to handle the complexity of the scene, and to synchronize the data stored in the different 'audioBuffer' nodes. Additionally, for each information a new stream has to be opened.

Method and apparatus for decoding a data stream in audio video streaming systems


A method, system, and computer product is presented for mapping a set of patterns into an m-dimensional space so as to preserve relationships that may exist between these patterns. A subset of the input patterns is chosen and mapped into the m-dimensional space using an iterative nonlinear mapping process based on subset refinements. A set of n attributes are determined for each pattern, and one or more neural networks or other supervised machine learning techniques are then trained in accordance with the mapping produced by the iterative process. Additional input patterns not in the subset are mapped into the m-dimensional space by determining their n input attributes and using the neural networks in a feed-forward (prediction) mode.

1. Field of the Invention
The present invention relates to information representation, information cartography and data mining. The present invention also relates to pattern analysis and representation, and, in particular, representation of object relationships in a multidimensional space.
2. Related Art
Similarity is one of the most ubiquitous concepts in science. It is used to analyze and categorize phenomena, rationalize behavior and function, and design new entities with desired or improved properties. It is employed in virtually all scientific and technical fields, and particularly in data mining and information retrieval. Similarity (or dissimilarity) is typically quantified in the form of a numerical index derived either through direct observation, or through the measurement of a set of characteristic attributes, which are subsequently combined in some form of similarity or distance measure. For large collections of objects, similarities are usually described in the form of a matrix that contains some or all of the pairwise relationships between the objects in the collection. Unfortunately, pairwise similarity matrices do not lend themselves for numerical processing and visual inspection. A common solution to this problem is to embed the objects into a low-dimensional Euclidean space in a way that preserves the original pairwise relationships as faithfully as possible. This approach, known as multidimensional scaling (MDS) (Torgeson, W. S., Psychometrika 17:401–419 (1952); Kruskal, J. B., Phychometrika 29:115–129 (1964)) or nonlinear mapping (NLM) (Sammon, J. W., IEEE Trans. Comp. C18:401–409 (1969)), converts the data points into a set of real-valued vectors that can subsequently be used for a variety of pattern recognition and classification tasks.
Multidimensional scaling originated in the field of mathematical psychology and has two primary applications: 1) reducing the dimensionality of high-dimensional data in a way that preserves the original relationships of the data objects, and 2) producing Cartesian coordinate vectors from data supplied directly in the form of similarities or proximities, so that they can be analyzed with conventional statistical and data mining techniques.
Given a set of k objects, a symmetric matrix, rij, of relationships between these objects, and a set of images on a m-dimensional display plane {yi, i=1, 2, . . . , k; yi∈ m}, the problem is to place yi onto the plane in such a way that their Euclidean distances dij=∥yi−yj∥ approximate as closely as possible the corresponding values rij. The quality of the projection is determined using a sum-of-squares error function such as Kruskal's stress:
                    S        =                                                            ∑                                  i                  <                  j                                                                                (                                                            d                      ij                                        -                                          r                      ij                                                        )                                2                                                                    ∑                                  i                  <                  j                                                                          r                ij                2                                                                        (        1        )            which is numerically minimized in order to find the optimal configuration. The actual embedding is carried out in an iterative fashion by: 1) generating an initial set of coordinates yi, 2) computing the distances dij, 3) finding a new set of coordinates yi using a steepest descent algorithm such as Kruskal's linear regression or Guttman's rank-image permutation, and 4) repeating steps 2 and 3 until the change in the stress function falls below some predefined threshold.
A particularly popular implementation is Sammon's nonlinear mapping algorithm (Sammon, J. W. IEEE Trans. Comp., 1969). This method uses a modified stress function:
                    E        =                                            ∑                              i                <                j                            k                                                                                [                                                            r                      ij                                        -                                          d                      ij                                                        ]                                2                                            r                ij                                                                        ∑                              i                <                j                            k                                                  r              ij                                                          (        2        )            which is minimized using steepest descent. The initial coordinates, yi, are determined at random or by some other projection technique such as principal component analysis, and are updated using Eq. 3:yij(t+1)=yij(t)−λΔij(t)  (3)where t is the iteration number and λ is the learning rate parameter, and
                                          Δ            ij                                          (            t            )                          =                                            ∂                              E                                                  (                  t                  )                                                                    ∂                                                y                  ij                                                                  (                  t                  )                                                                                                                                      ∂                  2                                                                  E                                                        (                    t                    )                                                                              ∂                                                                            y                      ij                                                                                (                      t                      )                                                        2                                                                                                    (        4        )
There is a wide variety of MDS algorithms involving different error functions and optimization heuristics, which are reviewed in Schiffman, Reynolds and Young, Introduction to Multidimensional Scaling, Academic Press, New York (1981); Young and Hamer, Multidimensional Scaling: History, Theory and Applications, Erlbaum Associates, Inc., Hillsdale, N.J. (1987); Cox and Cox, Multidimensional Scaling, Number 59 in Monographs in Statistics and Applied Probability, Chapman-Hall (1994), and Borg, I., Groenen, P., Modern Multidimensional Scaling, Springer-Verlag, New York, (1997). The contents of these publications are incorporated herein by reference in their entireties.
Unfortunately, the quadratic nature of the stress function (Eqs. 1 and 2, and their variants) make these algorithms impractical for large data sets containing more than a few hundred to a few thousand items. Several attempts have been devised to reduce the complexity of the task. Chang and Lee (Chang, C. L., and Lee, R. C. T., IEEE Trans. Syst., Man, Cybern., 1973, SMC-3, 197–200) proposed a heuristic relaxation approach in which a subject of the original objects (the frame) are scaled using a Sammon-like methodology, and the remaining objects are then added to the map by adjusting their distances to the objects in the frame. An alternative approach proposed by Pykett (Pykett, C. E., Electron. Lett., 1978, 14, 799–800) is to partition the data into a set of disjoint clusters, and map only the cluster prototypes, i.e. the centroids of the pattern vectors in each class. In the resulting two-dimensional plots, the cluster prototypes are represented as circles whose radii are proportional to the spread in their respective classes. Lee, Slagle and Blum (Lee, R. C. Y., Slagle, J. R., and Blum, H., IEEE Trans. Comput., 1977, C-27, 288–292) proposed a triangulation method which restricts attention to only a subset of the distances between the data samples. This method positions each pattern on the plane in a way that preserves its distances from the two nearest neighbors already mapped. An arbitrarily selected reference pattern may also be used to ensure that the resulting map is globally ordered. Biswas, Jain and Dubes (Biswas, G., Jain, A. K., and Dubes, R. C., IEEE Trans. Pattern Anal. Machine Intell., 1981, PAMI-3(6), 701–708) later proposed a hybrid approach which combined the ability of Sammon's algorithm to preserve global information with the efficiency of Lee's triangulation method. While the triangulation can be computed quickly compared to conventional MDS methods, it tries to preserve only a small fraction of relationships, and the projection may be difficult to interpret for large data sets.
The methods described above are iterative in nature, and do not provide an explicit mapping function that can be used to project new, unseen patterns in an efficient manner. The first attempt to encode a nonlinear mapping as an explicit function is due to Mao and Jain (Mao, J., and Jain, A. K., IEEE Trans. Neural Networks 6(2):296–317 (1995)). They proposed a 3-layer feed-forward neural network with n input and m output units, where n and m are the number of input and output dimensions, respectively. The system is trained using a special back-propagation rule that relies on errors that are functions of the inter-pattern distances. However, because only a single distance is examined during each iteration, these networks require a very large number of iterations and converge extremely slowly.
An alternative methodology is to employ Sammon's nonlinear mapping algorithm to project a small random sample of objects from a given population, and then "learn" the underlying nonlinear transform using a multilayer neural network trained with the standard error back-propagation algorithm or some other equivalent technique (see for example, Haykin, S. Neural Networks: A Comprehensive Foundation. Prentice-Hall, 1998). Once trained, the neural network can be used in a feed-forward manner to project the remaining objects in the plurality of objects, as well as new, unseen objects. Thus, for a nonlinear projection from n to m dimensions, a standard 3-layer neural network with n input and m output units is used. Each n-dimensional object is presented to the input layer, and its coordinates on the m-dimensional nonlinear map are obtained by the respective units in the output layer (Pal, N. R. Eluri, V. K., IEEE Trans. Neural Net., 1142–1154 (1998)).
The distinct advantage of this approach is that it captures the nonlinear mapping relationship in an explicit function, and allows the scaling of additional patterns as they become available, without the need to reconstruct the entire map. However, as it was originally proposed, the method can only be used for dimension reduction, and requires that the input patterns be supplied as real vectors.
Hence there is a need for a method that can efficiently process large data sets, e.g., data sets containing hundreds of thousands to millions of items, and can be used with a wide variety of pattern representations and/or similarity distance functions. Moreover, there is a need for a method that is incremental in nature, and allows the mapping of new samples as they become available, without the need to reconstruct an entire map.

Method, system, and computer program product for representing object relationships in a multidimensional space

Johnson & Johnson Pharmaceutical Research & Development, L.L.C.


A method of detecting a substance includes, for each position of a plurality of positions in a field of view of an environment: emitting from a light source a light beam including a predetermined wavelength that is absorbable by a constituent of the substance, controlling at least one mirror to direct the emitted light beam to the position in the field of view, detecting by a detector light resulting from the emitted light beam, and determining whether the constituent of the substance is present at the position in the field of view based on characteristics of the detected light; generating a detection map indicating a presence or an absence of the substance at the plurality of positions in the field of view of the environment; capturing, by an image capture device, an image of the environment; and identifying a portion of the captured image having the substance based on the detection map.

Substance detectors and video cameras are both beneficial for various reasons. Substance detectors, such as vape or smoke detectors, can identify harmful activities or situations and provide an alert when there is a detection. While substance detectors are beneficial for detection, they do not identify the source of the harmful activity or situation. Video cameras can be used for surveillance and for identification. However, video cameras do not detect harmful activities or situations. Accordingly, there is interest in developing improved detection and surveillance systems.

Systems and methods for mapping absorption spectroscopy scans and video frames