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Patent application title: INHIBITION-BASED HIGH-THROUGHPUT SCREEN STRATEGY FOR CELL CLONES
Wei-Kuang Liu (Taipei City, TW)
Min-Pey Ding (Taipei, TW)
ScinoPharm Taiwan Ltd.
IPC8 Class: AC40B3000FI
USPC Class:
506 7
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library
Publication date: 2010-12-16
Patent application number: 20100317532
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A method for screening cells with high level expression of a target
protein is disclosed. The method includes introducing into a plurality of
host cells a DNA construct that encodes both a target protein and an
inhibitor to an endogenous selectable marker in the host cells, screening
host cells harboring the DNA construct for the expression of the
endogenous selectable marker, and isolating cells with reduced expression
of the selectable marker. Also disclosed is a DNA construct configured to
express both the target protein and the inhibitor inside the host cell.
1. A method for screening cells with high level expression of a target
protein, comprising:introducing into a plurality of host cells a DNA
construct that encodes both a target protein and an inhibitor to an
endogenous selectable marker in the host cells, wherein the DNA construct
is configured to express both the target protein and the inhibitor inside
the host cell;screening host cells harboring the DNA construct for the
expression of the endogenous selectable marker, andisolating cells with
reduced expression of the selectable marker.
2. The method of claim 1, wherein the inhibitor is selected from the group consisting of small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), hybrid of miRNA and shRNA, and antisense RNA.
3. The method of claim 1, wherein the inhibitor is a shRNA.
4. The method of claim 1, wherein the endogenous selectable marker is a fluorescent marker and wherein the isolating step comprises sorting cells with a fluorescence activated cell sorter (FACS).
5. The method of claim 1, wherein the DNA construct further encodes a dihydroforate reductase (DHFR) and wherein the host cells are DHFR-deficient cells.
6. A high throughput screening method for selecting transgene expressing cells, comprising:transfecting the cells with a vector carrying at least one transgene and an interfering RNA that inhibits the expression of the fluorescent protein;measuring fluorescence intensity in the transfected cells; andisolating cells having a fluorescence intensity that is lower than the fluorescence intensity of untransfected cells.
7. The method of claim 6, wherein the fluorescent protein is green fluorescent protein (GFP).
8. The method of claim 6, wherein the interfering RNA is a mir-30-based shRNA.
9. The method of claim 6, wherein the isolating step comprises sorting cells with a FACS.
10. The method of claim 6, wherein the cells that express a fluorescent protein are dihydroforate reductase (DHFR)-deficient CHO cells.
11. The method of claim 6, wherein the at least one transgene is linked to a gene encoding DHFR by an internal ribosome entry site (IRES).
12. An expression vector for high-throughput screening of cells harboring the expression vector, comprising:a first nucleotide sequence encoding a target protein;a second nucleotide sequence encoding an exogenous selection marker for a host cell;a third nucleotide sequence encoding an inhibitor to an endogenous selection marker in the host cell; andone or more regulatory elements that control the expression of the first, second and third nucleotide sequences in the host cell,wherein the first nucleotide sequence is linked to the second nucleotide sequence by an internal ribosome entry site (IRES).
13. The expression vector of claim 12, further comprising one or more anti-repressor elements.
14. The expression vector of claim 13, wherein the one or more anti-repressor elements includes a partial mouse anti-repressor element 40.
15. The expression vector of claim 12, wherein the inhibitor is an interfering RNA.
16. The expression vector of claim 15, wherein the interfering RNA is a miR-30-based shRNA.
17. The expression vector of claim 12, wherein the endogenous selection marker is a fluorescent protein.
18. The expression vector of claim 17, wherein the fluorescent protein is green fluorescent protein.
19. The expression vector of claim 12, wherein the exogenous selectable marker is dihydroforate reductase.
20. The expression vector of claim 12, wherein the one or more regulatory elements include a CMV IE enhancer.
[0001]This application claims the priority of U.S. Provisional Application No. 61/213,459, filed on Jun. 11, 2009, which is incorporated herein by reference in its entirety.
[0002]The disclosed technology generally relates to biotechnology and molecular biology and, in particular, to high-throughput screening of cell clones.
[0003]A key step in the generation of cell lines producing recombinant proteins is the selection of viable clones following the incorporation of the gene of interest into the host cell. For industrial-scale bio-production, a clone in which the product gene is stably integrated and yields a high level of the protein product is highly desired.
[0004]In a heterogeneous population, the integration of a recombinant gene into the host genome is a largely random event. The proportion of cells containing multiple copies of stably integrated genes would be small compared to those with low copy numbers. The high-producing subclones are rare and tend to be diluted out by the faster growing non- or low-producing cells. Thus, to isolate a subclone with an increased production rate, many wells would need to be screened and tested. In general, limited dilution methods are tedious and time consuming.
[0005]The advent of selection methods that use flow cytometry and cell sorting considerably increased the number of cells that can be screened. Several million cells can be screened in a short time, and subpopulations and single cells can be isolated from within mixed-cell populations even when they are present at frequencies as low as 10-6 within the population.
[0006]Flow cytometry was partnered with a non-fluorescent reporter protein for rapid, early stage identification of clones producing high levels of a target protein. This has been facilitated by the availability of antibody and ligand-conjugated fluorochromes enabling isolation of cells based on cell-surface protein expression. For example, a cell surface protein, not normally expressed on host cells, may be co-expressed, with the target protein as a reporter.
[0007]In the absence of a correlation between surface expression and productivity, cells can be isolated based on levels of intracellular proteins using reporter molecules such as green fluorescent protein (GFP). The GFP has become an important reporter for gene expression and the selection of cells based on inducible gene products. In mammalian cell lines, GFP has been used for the selection of high-producing clones by co-expression with recombinant proteins and selection based on fluorescence intensity. A correlation between GFP fluorescence intensity and recombinant protein production has been seen for several cell lines expressing various recombinant proteins. However, expression of these selection markers increases the load on the protein expression machinery in the cells and reduces the production of the target protein.
[0008]One aspect of the present invention relates to a method for screening cells with high level expression of a target protein. The method includes introducing into a plurality of host cells a DNA construct that encodes both a target protein and an inhibitor to an endogenous selectable marker in the host cells, screening host cells harboring the DNA construct for the expression of the endogenous selectable marker, and isolating cells with reduced expression of the selectable marker. The DNA construct is configured to express both the target protein and the inhibitor inside the host cell.
[0009]In an embodiment, the inhibitor is selected from the group consisting of small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), hybrid of miRNA and shRNA, and antisense RNA.
[0010]In another embodiment, the inhibitor is a shRNA.
[0011]In another embodiment, the endogenous selectable marker is a fluorescent marker and the isolating step comprises sorting cells with a fluorescence activated cell sorter (FACS).
[0012]In another embodiment, the DNA construct further encodes a dihydroforate reductase (DHFR) and the host cells are DHFR-deficient cells.
[0013]Another aspect of the present invention relates to a high throughput screening method for selecting transgene expressing cells. The method includes transfecting host cells that express a fluorescent protein with a vector carrying at least one transgene and an interfering RNA that inhibits the expression of the fluorescent protein; measuring fluorescence intensity in the transfected cells; and isolating cells having a fluorescence intensity that is lower than the fluorescence intensity of untransfected cells.
[0014]In one embodiment, the fluorescent protein is green fluorescent protein (GFP).
[0015]In another embodiment, the interfering RNA is a mir-30-based shRNA.
[0016]In another embodiment, the isolating step comprises sorting cells with a FACS.
[0017]In another embodiment, the cells that express a fluorescent protein are DHFR-deficient CHO cells.
[0018]In another embodiment, the at least one transgene is linked to a gene encoding DHFR by an internal ribosome entry site (IRES).
[0019]Another aspect of the present invention relates to an expression vector for high-throughput screening of cells harboring the expression vector. The expression vector comprises a first nucleotide sequence encoding a target protein, a second nucleotide sequence encoding an exogenous selection marker for a host cell, a third nucleotide sequence encoding an inhibitor to an endogenous selection marker in the host cell, and one or more regulatory elements that control the expression of the first, second and third nucleotide sequences in the host cell. The first nucleotide sequence is linked to the second nucleotide sequence by an internal ribosome entry site (IRES).
[0020]In an embodiment, the expression vector further comprises one or more anti-repressor elements.
[0021]In a related embodiment, the one or more anti-repressor elements includes a partial mouse anti-repressor element 40.
[0022]In another embodiment, the inhibitor is an interfering RNA.
[0023]In a related embodiment, the interfering RNA is a miR-30-based shRNA.
[0024]In another embodiment, the endogenous selection marker is a fluorescent protein.
[0025]In a related embodiment, the fluorescent protein is green fluorescent protein.
[0026]In another embodiment, the exogenous selectable marker is dihydroforate reductase.
[0027]In another embodiment, the one or more regulatory elements include a CMV IE enhancer.
[0028]FIG. 1 is a diagram showing a GFP-based screening strategy for high transgene expression cell clones.
[0029]FIG. 2 is a map of expression vector pScinoDP-DHFR.
[0030]FIG. 3 is a map of expression vector pScinoDP3-DHFR.
[0031]FIG. 4 is a map of expression vector pScinoDP3mir-DHFR.
[0032]FIG. 5 is a map of expression vector pScinoDP8mir-DHFR.
[0033]FIG. 6 is a map of expression vector pScinoDP9mir-DHFR.
[0034]FIGS. 7A and 7B are diagrams showing the construction of expression vector pScinoDP9mir-Herceptin-DHFR.
[0035]FIG. 8 is a composite of diagrams showing GFP expression and antibody expression in herceptin-CHO+GFP/-dhfr cells. Panel A: FACS histogram profile of herceptin-CHO.sup./+GFP/-dhfr cells. Panels B-C: FACS histogram profiles of herceptin-CHO.sup./+GFP/-dhfr cell sorted at different fluorescence intensity levels. Panel E: ELISA analysis of antibody production of cells sorted from different fluorescent populations.
[0036]FIG. 9 is a composite of diagrams showing GFP expression in herceptin-CHO.sup./+GFP/-dhfr cells after multiple rounds of selection. Panel A: FACS histogram profile of CHO/-dhfr cells after GFP expression. Panel B: FACS histogram profile of herceptin-CHO.sup./+GFP/-dhfr cells after G418 selection. Panel C: FACS histogram profile of herceptin-CHO.sup./+GFP/-dhfr cells after first round of MTX (100 nM) amplification. Panel D: FACS histogram profile of herceptin-CHO.sup./+GFP/-dhfr cells after second round of MTX (200 nM) amplification.
[0037]FIG. 10 is a diagram showing FACS analysis of herceptin-CHO+GFP/-dhfr cells stained with a PE-conjugated anti-human IgG(Fc) antibody. Cells secreting antibodies were PE positive and GFP negative according to experimental design and shown in upper-left panel. Fluorescence signal was determined by FACS and calibrated with standard compensation protocol.
[0038]FIG. 11 shows the key features of the FACS sorting procedure for herceptin-CHO+GFP/-dhfr cells and ELISA results. Panel A: FACS histogram profile for Herceptin (CHO/+GFP/-dhfr) cells. Horizontal bars and gray regions indicate the low-fluorescence and high-fluorescence cell populations used for the single cell analysis. Panel B: Antibody expression levels detected by ELISA of each individual clones selected from low-fluorescence or high-fluorescence populations.
[0039]FIG. 12 is a composite of diagrams showing FACS analysis of GFP expression in cells transfected with DNA constructs encoding shRNAGFP and Herceptin.
[0040]A method for screening cells with high level expression of an exogenous protein is disclosed. In one embodiment, the method includes the steps of introducing into a plurality of host cells a DNA construct that encodes both a target protein and an inhibitor to an endogenous selectable marker in the host cells, wherein the construct is configured to express both the target protein and the inhibitor inside the host cell; screening host cells harboring the DNA construct for the expression of the endogenous selectable marker, and isolating cells with reduced expression of the endogenous selectable marker.
[0041]As used hereinafter, the terms "cell"/"host cell" and "cell line"/"host cell line" are respectively typically defined as a eukaryotic cell and homogeneous populations thereof that are maintained in cell culture by methods known in the art, and that have the ability to express heterologous proteins. In one embodiment, the cells are CHO cells. In another embodiment, the cells are CHO cells that express GFP as an endogenous selection marker. In another embodiment, the cells are CHO cells that are deficient in DHFR gene and express GFP as an endogenous selection marker.
[0042]As used hereinafter, the term "expression" is typically used to refer to the production of a specific RNA product or products, or a specific protein or proteins, in a cell. In the case of RNA products, it refers to the process of transcription. In the case of protein products, it refers to the processes of transcription, translation and optionally post-translational modifications. In the case of secreted proteins, it refers to the processes of transcription, translation, and optionally post-translational modification (e.g., glycosylation, disfulfide bond formation, etc.), followed by secretion. In the case of multimeric proteins, it includes assembly of the multimeric structure from the polypeptide monomers. The corresponding verbs of the noun "expression" have an analogous meaning as the noun.
[0043]As used hereinafter, the term "selectable marker" is typically used to refer to a gene and/or protein whose presence can be detected directly or indirectly in a cell, for example, a gene and/or a protein that inactivates a selection agent and protects the host cell from the agent's lethal or growth-inhibitory effects (e.g., an antibiotic resistance gene and/or protein). Another possibility is that the selection marker induces fluorescence or a color deposit (e.g., green fluorescent protein and derivatives, luciferase, or alkaline phosphatase). The term "endogenous selectable marker" refers to a selectable marker that is encoded by a polynucleotide that is present in the host cell prior to the introduction of the DNA construct into the host cell. The coding sequence for the "endogenous selectable marker" may exist in either integrated form (i.e., integrated into the cell genome) or in episomal form.
[0044]As used hereinafter, the term "DNA construct" refers to an expression or transformation construct. The DNA construct comprises at least one expression unit or expression cassette. The term "expression unit or expression cassette" is herein defined as a unit capable of expressing a coding sequence or an open reading frame. An "expression unit or expression cassette" typically comprises one or more regulatory elements operably linked to a transgene that encodes a molecule of interest (i.e., a polypeptide or a polynucleotide).
[0045]A "regulatory element" is a nucleic acid sequence that regulates the expression of a transgene by being operably linked to the coding sequence. Examples of regulatory sequences include, but are not limited to, appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. A regulatory sequence may act in cis or trans configuration, or at a distance to control a gene of interest.
[0046]A "transgene" is a nucleic acid sequence that is to be delivered or transferred to a mammalian cell. A transgene may encode a protein, peptide or polypeptide that is useful as a marker, reporter or therapeutic molecule. A transgene may also encode a protein, polypeptide or peptide that is useful for protein production, diagnostic assays or for any transient or stable gene transfer in vitro or in vivo. Alternatively, a transgene may encode a functional polynucleotide, such as miRNA, RNAi, shRNA, antisense RNAs, ribozyme or other regulatory nucleic acids. Transgenes also include DNA sequences that are used to induce DNA recombination and gene repair.
[0047]A nucleic acid sequence is "operably linked" to another nucleic acid sequence when the former is placed into a functional relationship with the latter. For example, a DNA for a presequence or secretory leader peptide is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the hepolypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
[0048]The "inhibitor to a selectable marker" can be a polypeptide or a polynucleotide that inhibits, directly or indirectly, the expression or activity of the selectable marker. In one embodiment, the inhibitor is a polynucleotide, such as a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a hybrid of miRNA and shRNA, or an antisense RNA molecule that inhibits the expression of the selectable marker in the host cell. In another embodiment, the inhibitor is a polypeptide, such as transcription regulator, that inhibits the expression of the selectable marker in the host cell. In yet another embodiment, the inhibitor is a polypeptide, such as an antibody, that inhibits a biological activity of the selectable marker.
[0049]As used herein, the term "siRNA" refers to an RNA agent, preferably a double-stranded agent, of about 10-50 nucleotides in length (the term "nucleotides" including nucleotide analogs), preferably between about 15-25 nucleotides in length, more preferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, the strands optionally having overhanging ends comprising, for example, 1, 2 or 3 overhanging nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. Naturally-occurring siRNAs are generated from longer dsRNA molecules (e.g., >25 nucleotides in length) by a cell's RNA interference (RNAi) machinery.
[0050]The term "RNA interference" or "RNAi", as used herein, refers generally to a sequence-specific or selective process by which a target molecule (e.g., a target gene, protein or RNA) is downregulated. In specific embodiments, the process of "RNA interference" or "RNAi" features degradation of RNA molecules, e.g., RNA molecules within a cell, said degradation being triggered by an RNA agent. Degradation is catalyzed by an enzymatic, RNA-induced silencing complex (RISC). RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by introducing small interfering RNA molecules into a cell to silence the expression of target genes.
[0051]As used hereinafter, the term "shRNA", refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it.
[0052]The DNA construct may further contain plasmid elements for replication and selection in bacteria during the construction of the DNA construct. The DNA construct may also contain other selection markers to facilitate the amplification of the DNA construct in the host cell. Selection marker(s) encoded by the DNA construct are considered exogenous selection markers. Examples of exogenous selection markers include, but are not limited to, resistance to antibiotics (e.g. neomycin gene encoding resistance to G418) or enzyme analogs (e.g. dihydrofolate reductase gene encoding methotrexate resistance).
[0053]In one embodiment, the DNA construct contains a bicistronic expression cassette in which the open reading frame for the protein of interest is linked to the coding sequence of the exogenous selection marker or the inhibitor of the endogenous marker by an internal ribosome entry site (IRES), so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the exogenous selection marker's or the inhibitor's expression level accurately predicts the relative expression level of the protein of interest for each clone. Preferably, the open reading frame for the protein of interest in the bicistronic expression cassette is located upstream of the coding sequence of the exogenous selection marker or the inhibitor of the endogenous selectable marker.
[0054]For example, to improve the accuracy and throughput of multiple rounds of methotrexate (MTX) amplification, the gene encoding a therapeutic protein (such as the light chain of an antibody) is linked to an exogenous selectable marker (such as DHFR) at the 3'-end by an IRES, so that they are transcribed in the same mRNA but are translated independently. The lower efficiency of IRES-mediated translation relative to 5' cap-mediated translation ensures that cellular resources are utilized mainly for production of the therapeutic protein rather than the DHFR protein. However, since they arise from the same mRNA, the DHFR amplification level accurately predicts, for each clone, the relative expression level of the therapeutic protein.
[0055]In another embodiment, the DNA construct further contains one or more anti-repressor element (ARE) that counteract chromatin associated repression. As used hereinafter, an ARE (or anti-repressor sequence, which is used interchangeably herein) is a naturally occurring DNA element isolated from eukaryotic genomes on the basis of its ability to block transgene repression. An ARE comprises the capacity to influence transcription of genes in cis and/or provide a stabilizing and/or an enhancing effect. It has been demonstrated that when AREs flank transgenes, the transgene expression level of randomly selected recombinant cell lines can be increased to levels approaching the maximum potential expression of the transgene's promoter. Moreover, the expression level of the transgene is stable over many cell generations, and does not manifest stochastic silencing. Therefore, ARE confers a degree of position-independent expression on transgenes that is not possible with conventional transgenic systems. The position independence means that transgenes that are integrated in genomic locations that would result in transgene silencing are, with the protection of ARE, maintained in a transcriptionally active state. In one embodiment, the ARE is a partial mouse ARE40 fragment. In another embodiment, the DNA construct contains multiple partial mouse ARE40 fragments.
[0056]Methods for introducing the DNA construct into the cell are well know in the art. Examples of such methods include, but are not limited to, electroporation, lipofection, calcium phosphate or calcium chloride co-precipitation and DEAE-dextran-mediated transfection. The DNA construct may also be introduced into the cells by a virus vector. Commonly used virus vectors include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors and retrovirus vectors.
[0057]Cells transfected with the DNA construct are screened for both the expression of the exogenous and endogenous selectable markers. High level expression of the selectable marker inhibitor from the DNA construct would result in reduced expression and/or activity of the endogenous selectable marker. Cells with reduced expression of the selectable marker are then isolated and subcloned to determine the level and stability of transgene expression.
[0058]In certain embodiments, the endogenous selectable marker is a protein that can be induced to emit fluorescence. In these embodiments, the screening step and the isolating step can be performed simultaneously using a fluorescence activated cell sorter (FACS).
[0059]In other embodiments, cells transfected with the DNA construct are first subjected to one or more rounds of selection with the exogenous marker (e.g., selection of G418 resistant and/or methotrexate resistant clones). The selected cells are then screened for reduced expression and/or activity of the endogenous selectable marker.
[0060]Also disclosed is a DNA construct configured to allow high-throughput screening of cells harboring the DNA construct. In one embodiment, the DNA construct contains coding sequences for a target protein and coding sequences for an inhibitor to an endogenous selectable marker in a host cell, wherein the DNA construct is configured to express both the target protein and the inhibitor inside the host cell.
[0061]In one embodiment, the inhibitor is a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a hybrid of miRNA and shRNA, or an antisense RNA molecule that inhibits the expression of the endogenous selectable marker in the host cell.
[0062]In another embodiment, the inhibitor is a polypeptide, such as transcription regulator, that inhibits the expression of the endogenous selectable marker in the host cell.
[0063]In another embodiment, the inhibitor is a polypeptide, such as an antibody, that inhibits a biological activity of the endogenous selectable marker.
[0064]In another embodiment, the endogenous selectable marker is a protein that induces fluorescence.
[0065]In another embodiment, the DNA construct further comprises one or more anti-repressor elements.
[0066]The technology described in the present invention enables rapid identification and isolation of high production clones from a heterogeneous population of transfected cells, decreasing the labor and time associated with standard limiting dilution cloning methods. In addition, because this technology can identify desired cells that are rare events in the population, it can shorten the development timeline by reducing the number of rounds of pool amplification prior to isolating the high producing clones or by first isolating the high producing clones for drug amplification and subcloning.
[0067]The ease and efficiency of green fluorescent protein (GFP)/fluorescence activated cell sorter (FACS)-based screening methods had been demonstrated in previous studies. In these previous reports, GFP was incorporated as part of a fusion protein or part of a bicistronic construct using either an internal ribosomal entry site (IRES) or a two promoter system. Cells with a high-level of GFP correlated with a high level of the protein product of interest. This can be attributed to the stable integration of a high number of copies of the recombinant gene or that the gene had been integrated into sites of very high transcriptional activity. Although these methods have been shown to be effective, there may be some concern regarding the use of GFP-containing cell lines for the production of human therapeutics. Furthermore, it appears unnecessary to burden the cell's metabolic machinery with GFP production after the subpopulation has been isolated. This cellular resource could potentially be diverted to increasing cell growth or recombinant protein production. The following examples describe a novel technique for selecting high-producing cell clones that reversely uses GFP as a selection marker, coupled with sorting through FACS.
[0068]FIG. 1 is a schematic showing the GFP-based selection process. Briefly, plasmid vectors containing the cDNA encoding for GFP are transfected into DHFR-deficient CHO cells. Cells that have successfully acquired the desired vector are fluorescent. The GFP-expression cell is set as the parental host cell for recombinant-protein expression.
[0069]For desired recombinant-protein production, the parental host cells are transfected with the shRNAmireGFP-containing vector, and then FACS sorted to screen for cells with low fluorescent intensity. Cell cultures after several rounds of increasing MTX challenge were subjected to repeated rounds of sorting and expansion. Cell clones with the lowest GFP fluorescence intensity would correspond to the clones with the highest transgene expression. Finally, the selected clones are expanded and tested for production and stability. As FACS enables a large number of cells to be easily screened, the chance of obtaining high producing clones would be greatly increased compared to limited dilution methods. Moreover, the procedure is less labor-intensive and may significantly reduce the time required to generate clones for bio-production.
Example 1
Establishment of CHO+GFP/-dhfr Cell Line
[0070]The Chinese Hamster Ovary dihydrofolate reductase deficient cell line (CHO/dhfr-) was maintained in Iscove's modified Dulbecco's medium (IMDM, Gibco, Cat. No. 12200) supplemented with HT (0.1 mM sodium hypoxanthine and 0.016 mM thymidine, Gibco, Cat No. 11067), 10% FBS (Biological Industries, Cat. 04-001-1A) and 2 μM Methothrexate Hydrate (MTX, Sigma, SI-M8407).
[0071]The CHO/dhfr- cells were transfected with 5 ug of plasmid vector containing the cDNA encoding for GFP (pFLAg-eGFP-IRES-Puro,) with lipofectamine according to the instructions from Lipofectamine® Invitrogen Puls® Reagent (Cat. No. 11514-015). Transfected cells were selected using 5 μg/ml of puromycin dihydrochloride (Sigma, SI-P8833). After 10 days of selection in IMDM medium supplemented with HT, 10% FBS, 2 μM MTX and selective antibiotics, cells were maintained in above medium. The GFP-expressing CHO-dhfr cell (CHO+GFP/-dhfr cell) is cloned and set as the parental host for recombinant-protein expression.
Example 2
Construction of Expression Vectors
[0072](1) The pScinoDP-DHFR Vector
[0073]The pScinoDP-DHFR plasmid was constructed by substituting the EGFP gene in the pEGFP-N1 (Clontech) plasmid backbone with an IRES-DHFR fused gene, and inserting an addition SV40 polyA tail and CMV-IE promoter into the pEGFP-N1 (Clontech) plasmid backbone. Briefly, polyA tail and CMV-IE promoter sequence were obtained by PCR amplification using pCEP4 plasmid (Invitrogen) as the template. A fused polyA tail-promoter fused sequence SV40polA-CMV-IE (about 1.2 kb) was created through overlap extension by PCR (OL-PCR). The fused sequence was digested with XhoI/BglII and inserted into XhoI/BglII digested pEGFP-N1 vector. The resulting construct was named pScinoDP.
[0074]IRES sequence and DHFR gene were obtained by PCR amplification using pIRES2-EGFP and pSV2-DHFR plasmids as template. A hybrid IRES sequence fragment and DHFR gene fragment (IRES2-DHFR) was produced through overlap extension by PCR (OL-PCR). The hybrid fragment (-1.1 kb) was digested with AgeI/NotI and inserted into AgeI/NotI digested pEGFP-N1 to substitute the EGFP gene in the pEGFP-N1 vector. The resulting construct was named pIRES2-DHFR. A site-directed mutagenesis was performed to eliminate the ApaLI site in the IRES sequence.
[0075]Plasmid pIRES2-DHFR, with mutated restricted enzyme ApaLI site in IRES sequence, was digested with AgeI/NotI and a 1.2 kb fragment containing IRES sequence and the entire DHFR coding region gene was ligated into AgeI/NotI-digested pScinoDP, generating pScinoDP-DHFR (FIG. 2). All constructs were confirmed by restriction analyses and/or by nucleotide sequencing.
(2) The pScinoDP3-DHFR Vector
[0076]The pScinoDP3-DHFR vector is a pScinoDP-DHFR based vector that carries a hEF1α promoter with the CMV-IE enhancer. Briefly, the hEF1α promoter was amplified from pBudCE4.1 vector (Invitrogen). Hybrid CMV-IE enhancer sequence with hEF1α promoter was obtained through sub-cloning hEF1α promoter after the CMV-IE enhancer on pEGFP-N1 to form pCMVe-hEF1α-EGFP vector. The CMVe-hEF1α fragment was PCR amplified from the pCMVe-hEF1α-EGFP vector and used to replace both CMV promoters in the pScionDP-DHFR vector. The resulting vector is named pScinoDP3-DHFR (FIG. 3).
(3) The pScinoDP3mir-DHFR Vector
[0077]The single strand 97 nt "mir30-like" shRNAiGFP oligo was generated using PCR as described using the following synthesis skeleton DNA and primers:
TABLE-US-00001 Single strand 97 nt "shRNAiGFP" DNA oligo (SEQ ID NO: 1) 5'TGCTGTTGACAGTGAGCGAGCACAAGCTGGAGTACAACTATAGT GAAGCCACAGATGTATAGTTGTACTCCAGCTTGTGCCTGCCTACTGC CTCGGA-3'
The underlined, italicized sequences represent the flanking mir30 sequences and the non-underlined, italicized sequence represents the mir30 loop structure. The sample sense and antisense-selected target sequences are shown in bold and underlined bold, respectively. The mir30-like shRNAiGFP is synthesized as a single stranded DNA oligonucleotide with common ends corresponding to part of the endogenous mir30 miRNA flanking sequence.
TABLE-US-00002 mirFWD-AgeI primer sequence (40 mers): (SEQ ID NO: 2) 5'-CAGAAGGACCGGTAAGGTATATTGCTGTTGACAGTGAGCG-3' mirREV-HindIII primer sequence (37 mers): (SEQ ID NO: 3) 5'-CTAAAGTAGCCCCTTAAGCTTTCCGAGGCAGTAGGCA-3'
[0078]The flanking regions, shown in underlined, italicized sequences, are used as universal flanks to prime a reaction, whereby the entire mir30-like shRNAiGFP is amplified to produce a PCR product that can be cloned into the recipient vector.
[0079]PCR was performed using Platinum® Pfx DNA Polymerase and the following profile: 95° C. for 3 min, then 95° C. for 30 s, 54° C. for 30 s, and 75° C. for 30 s for a total of 35 cycles. The resulting PCR products (AgeI-shRNAiGFP) were cloned into modified pEGFP-N1 vector (AgeI site destroyed, and additional AgeI and EcoRV sites behind Neomycin gene). The resulting construct was named pEGFP-N-1-shRNAiGFP. These AgeI-shRNAiGFP sequences were also confirmed by DNA sequencing.
[0080]The pEGFP-N-1-shRNAiGFP vector was digested with ApaLI-NotI. The CMV-IE-GFP fragment was replaced with the ScinoDP3-DHFR fragment from pScinoDP3-DHFR. The resulting construct was named pScinoDP3mir-DHFR (FIG. 4).
(4) The pScinoDP8mir-DHFR Vector
[0081]The pScinoDP8mir-DHFR vector contains a regulatory DNA element mARE40. Regulatory elements, such as anti-repressing element derived from housekeeping genes, were shown to positively affect specific productivities of recombinant proteins produced from cell lines. Plasmid pEGFP-N1 was used as backbone for this construct. Briefly, the partial mouse anti-repressor element 40 fragment was generated using overlapping-PCR as described using the following synthesis skeleton DNA and primers:
TABLE-US-00003 mARE40-L1(+) skeleton DNA: (SEQ ID NO: 4) 5'-TTGCTCTGAGCCAGCCCACCAGTTTGGAATGACTCCTTTTTATG ACTTGAATTTTCAAGTATAAAGTCTAGTGCTAAATTTAATTTGAACA ACTGTATAGTTTTTG-3' mARE40-L1(-) skeleton DNA: (SEQ ID NO: 5) 5'-TTAGAAATCCTCACACACAACAAGTTTTCATTTCACTTCTAATT CTGAAAAAAACACTGCCACCATTTTTTTTCCTTCCCCCAACCAGCAA AAACTATACAGTTGT-3' mARE40-R1(+) skeleton DNA (SEQ ID NO: 6) 5'-GTGTGTGAGGATTTCTAATGACATGTGGTGGTTGCATACTGAGT GAAGCCGGTGAGCATTCTGCCATGTCACCCCCTCGTGCTCAGTAATG TACTTTACAGAAATC-3' mARE40-R1(-) skeleton DNA (SEQ ID NO: 7) 5'-TGGCAGAAATGCAGGCTGAGTGAGACTACCCAGAGAAGAGACCG GATATACACAAGAAGCATGGTTTATATCAATCTTTTGAGTTTAGGAT TTCTGTAAAGTACAT-3' mARE40-5'primer: (SEQ ID NO: 8) 5'-TTGCTCTGAGCCAGCCCACCAGTTT-3' mARE40-3'AseI primer: (SEQ ID NO: 9) 5'-GTTATTAATTGGCAGAAATGCAGGCTGAGT-3' mARE40-3'AflII primer: (SEQ ID NO: 10) 5'-CCCACATGTTGGCAGAAATGCAGGCTGAGT-3' mARE40-3'SpeI primer: (SEQ ID NO: 11) 5'-GGACTAGTTGGCAGAAATGCAGGCTGAGTG-3'
[0082]PCR was performed using Platinum® Pfx DNA Polymerase and the following profile: 95° C. for 3 min, then 95° C. for 30 s, 58° C. for 30 s, and 75° C. for 30 s for a total of 35 cycles. The resulting PCR products (mARE40-AseI and mARE40-AflII) were separately cloned into modified pEGFP-N1 (AgeI site destroyed, and additional EcoRV before AseI, and ScaI site before AflII site) vector to form pmARE40-EGFP-N1, and (mARE40-SpeI) were cloned into the pScinoDP3-DHFR (additional EcoRV site before SpeI site) vector to form pScinoDP3-DHFR-F2. The pmARE40-EGFP-N1 vector was digested with AseI-BamHI and the CMV-IE promoter was replaced with the ˜1.6 kb AseI-CMVe-hEF1α-BamHI fragment from pScinoDP3-DHFR vector. The resulting construct was named pFmARE40ScinoDP3-EGFP-N1. The pFmARE40ScinoDP3-EGFP-N1 vector was digested with BamHI-NotI and the EGFP gene was replaced with the ˜1.6 kb BamHI-SV40polA-mARE40-DP3-IRES-DHFR-NotI fragment from pScinoDP3-DHFR-F2. The resulting construct was named pScinoDP8-DHFR. The pEGFP-N-1-shRNAiGFP vector was then digested with ApaLI-NotI and the CMV-IE-GFP fragment was replaced with the ScinoDP8-DHFR fragment from pScinoDP8-DHFR. The resulting construct was named pScinoDP8mir-DHFR (FIG. 5). The complete sequence of the cloned partial mouse anti-repressor element 40 fragment is shown below:
TABLE-US-00004 (SEQ ID NO: 12) 5'-TTgCTCTgAgCCAgCCCACCAgTTTggAATgACTCCTTTTTATg ACTTgAATTTTCAAgTATAAAgTCTAgTgCTAAATTTAATTTgAACA ACTgTATAgTTTTTgCTggTTgggggAAggAAAAAAAATggTggCAg TgTTTTTTTCAgAATTAgAAgTgAAATgAAAACTTGTTgTgTgTgAg gATTTCTAATgACATgTggTggTTgCATACTgAgTgAAgCCggTgAg CATTCTgCCATgTCACCCCCTCgTgCTCAgTAATgTACTTTACAgAA ATCCTAAACTCAAAAgATTgATATAAACCATgCTTCTTgTgTATATC CggTCTCTTCTCTGGGTAgTCTCACTCAgCCTgCATTTCTgCCA-3'.
(5) The pScinoDP9mir-DHFR Vector
[0083]The pScinoDP9mir-DHFR vector (FIG. 6) was constructed using procedures similar to those used for the construction of pScinoDP8mir-DHFR vector and substituting the two CMV enhancers in pScinoDP8mir-DHFR with CAG promoter (CMV-IE enhancer fused with chicken β-actin promoter). The complete sequence of pScinoDP9mir-DHFR is shown in SEQ ID NO:13.
[0084]pScinoDP9mir-DHFR contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) between the MCSII and the dihydrofolate reductase (DHFR) coding region. This permits both the gene of interest (for example: light chain cloned into the MCSII) and the DHFR gene to be translated from a single bicistronic mRNA. Sequences flanking DHFR have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. The MCSI in pScinoDP9mir-DHFR is between the immediate early promoter of CMV (PCMV IE) and SV40 polyadenylation signals sequences. SV40 polyadenylation signals downstream of the MCSI direct proper processing of the 3' end of the first transcription. The MCSII in pScinoDP-dhfr is between the second immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the DHFR gene direct proper processing of the 3' end of the bicistronic mRNA.
[0085]Because pScinoDP9mir-DHFR is derived from pEGFP-N1 vector, it contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pScinoDP-DHFR backbone also contains a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
(6) The pGFP/Puromycin Vector
[0086]IRES sequence and DHFR gene were obtained by PCR amplification used pIRES2-EGFP and pLKO-AS3w-puro plasmid as template. A hybrid IRES sequence fragment and puromycin gene fragment (IRES2-puromycin) was obtained through overlap extension by PCR (OL-PCR). The IRES2-puromycin fragment was inserted into SalI-BamHI digested pFLAG-CMV2 vector (Kodak). The resulting construct was named pIRES2-Puro. The EGFP gene was obtained from the pEGFP-N1 vector and inserted into the pIRES-Puro vector to form pGFP/Puromycin vector.
(7) The pScinoDP9mir-Herceptin-DHFR Vector
[0087]The construction of the pScinoDP9mir-Herceptin-DHFR vector is shown in FIGS. 7A and 7B. Briefly, the 4-1Leader sequence and 4-2Leader sequence were PCR amplified from oligo-synthesis skeleton fragments, while heavy chain constant region sequence of human IgG1(hIgG1CH) and light chain constant region sequence of human IgG1(hIgG1CL) were obtained from recombinant plasmids containing human IgG1 sequence. Hybrid leader sequence and hIgG1 constant region sequence vectors were obtained by sub-cloning hIgG1 constant region to leader sequence containing vector by the way of orientation linkage.
[0088]Next, variant region of heavy chain (VH) and light chain (VL) were created through repeated overlapping PCR from oligo-synthesis skeleton fragments and sub-cloned into p4-1Leader-hIgG1CH or p4-2Leader-hIgG1CL vector. After correcting PCR errata and removing additional sequences introduced during cloning process, the correctness of the leader-peptide-HerceptinVC sequence was verified by sequencing (FIG. 7A).
[0089]Finally, the hybrid 4-1Leader-Herceptin heavy chain (4-1-HerceptinVCH), and 4-2Leader-Herceptin light chain (4-2-HerceptinVCL) were individually sub-cloned into the MCSI and MCSII sites in pScinoDP9mir-DHFR vector to form the pScinoDP9mir-Herceptin-DHFR vector (FIG. 7B).
[0090]The amino acid and nucleotide sequences for the variant region of anti-HER2 heavy chain 1 are shown in SEQ ID NOS: 14 and 15, respectively. The skeleton fragments and PCR primers used for the variant region of the heavy chain (VH) have the following sequences:
TABLE-US-00005 Skeleton fragments: HerceptinVH-L1(+) (108 mer) (SEQ ID NO: 16) 5'-gAggTgCAgCTCgTggAgAgTggTggCgggTTggTCCAgCCAgg CgggTCTCTgCgATTgAgCTgTgCTgCCTCTggATTTAACATCAAAg ACACgTACATCCATTgg-3' HerceptinVH-L2(-) (105 mer) (SEQ ID NO: 17) 5'-TTTAACgCTATCAgCgTATCTggTgTAgCCgTTAgTgggATAgA TTCTAgCTACCCATTCAAggCCCTTgCCgggggCCTgTCTCACCCAA TggATgTACgTgTC-3' HerceptinVH-R1(+) (105 mer) (SEQ ID NO: 18) 5'-TACgCTgATAgCgTTAAAggAAggTTTACTATTTCTgCCgACAC CTCCAAgAATACCgCATATCTACAgATgAACTCCCTgCgCgCTgAgg ACACCgCTgTgTAT-3' HerceptinVH-R2(-) (108 mer) (SEQ ID NO: 19) 5'-CTTAgTAgAgCACTgCTAACTgTCACTAAggTACCCTggCCCCA gTAgTCCATTgCgTAgAATCCgTCTCCCCCCCAACgTgAgCAgTAAT ACACAgCggTgTCCTC-3' Primers for Amplification: Herceptin-VH-5HindIII (30 mer) (sense) (SEQ ID NO: 20) 5'-gCCAAgCTTgAggTgCAgCTCgTggAgAgT-3' Herceptin-VH-3ApaI (30 mer) (antisense) (SEQ ID NO: 21) 5'-AgggggCCCTTAgTAgAggCACTgCTAACT-3'
[0091]The amino acid and nucleotide sequences for the variant region of anti-HER2 light chain 1 are shown in SEQ ID NOS:22 and 23, respectively. The skeleton fragments and PCR primers used for the variant region of the light chain (VL) have the following sequences:
TABLE-US-00006 Skeleton Fragments: HerceptinVL-L1(+) (93 mer) (SEQ ID NO: 24) 5'-gATATACAgATgACACAgTCTCCgTCAAgTCTgAgCgCAAgCgT gggCgACCggGTAACAATTACCTgTAgAgCCAgCCAggACgTAAATA CA-3' HerceptinVL-L2(-) (95 mer) (SEQ ID NO: 25) 5'-CCgCTATAAAggAACgAggCAgAgTAgATCAgAAgCTTAggAGC TTTACCAggTTTTTgCTgATACCAggCCACggCTgTATTTACgTCCT ggCT-3' HerceptinVL-R1(+) (94 mer) (SEQ ID NO: 26) 5'-CTCgTTCCTTTATAgCggggTgCCAAgCCgCTTCTCCggATCTA ggTCTggAACAgACTTTACTCTgACCATTTCCAgTCTCCAgCCCgAA gAC-3' HerceptinVL-R2(-) (93 mer) (SEQ ID NO: 27) 5'-CTTgATCTCgACCTTggTgCCCTgCCCAAATgTgggTggAgTCg TgTAATgTTgCTggCAATAgTAggTAgCAAAgTCTTCgggCTggAgA CT-3' Primers for Amplification Herceptin-VL-5HindIII (30 mer) (sense) (SEQ ID NO: 28) 5'-gCCAAgCTTgATATACAgATgACACAgTCT-3' Herceptin-VL-3BamHI (30 mer) (antisense) (SEQ ID NO: 29) 5'-CgCggATTCCTTgATCTCgACCTTggTgCC-3'
Example 3
Establishment of Herceptin/CHO+GFP/-dhfr Cell Line
[0092]CHO.sup./+GFP/-dhfr cells were suspended in PBS buffer. 40 μg of linearized plasmid (pScinoDP9mir-Herceptin-DHFR) DNA was added to the cells and incubated on ice for 10 min. The cells were then electroported by two pulses at a voltage setting of 750 V and a capacitance setting at 25 μF (Gene Pulser II with capacitance extender, and pulse controller from Bio-Rad). The electroporated cells were plated in T-175 flask with 25 mL of medium (IMDM supplemented with HT, 10% FBS, 2 μM MTX and 5 μg/ml puromycin dihydrochloride) for 24 hours. The cells were then selected using 800 μg/ml of G418 sulfate (Calbiochem, Cat# 345810) in Minimum Essential medium Alpha medium (Alpha-MEM, Gibco, Cat. No. 12000) supplemented with 10% D-FBS (Gibco, Cat. No. 30067-334) and 5 μg/ml of puromycin dihydrochloride. After 14 days of selection in Alpha-MEM medium supplemented with 10% D-FBS and selective antibiotics, the cells were subjected to MTX treatment at increasing concentrations for gene amplification.
[0093]The cells were FACS sorted to screen for cells with low GFP fluorescent intensity. Since the expression of the target gene and shRNAGFP would lead to reduced GFP production in the CHO+GFP/-dhfr cells, cells with least green fluoresence (GFP negative) would have the highest level of target gene expression. FACS was performed using a MoFlo® XDP (Beckman Coulter) equipped with Summit software, an laser emitting at 488 nm and a cell deposition unit for sorting.
[0094]Low-fluorescence and high-fluorescence cell populations were sorted with the single cell deposited using FACS into 96-well cell culture plates containing 220 μl Alpha-MEM supplemented with 10% D-FBS, G418, puromycin dihydrochloride and MTX. Clones were incubated at 37° C. and 5% carbon dioxide in a humidified incubator for 12 days.
Example 4
Characterization of Herceptin/CHO+GFP/-dhfr Cell Line
[0095]Trypsinized herceptin/CHO+GFP/-dhfr cells were centrifuged 5 min at 200 rpm. The cells were washed twice with PBS and resuspended in PBS to a final concentration of about 1×107 cells/ml. The cells were then incubated with phycoerythrin (PE)-conjugated mouse anti-human IgG(Fc) (Beckman Coulter, Cat. No. 736007) at dilutions according to the manufacturer's recommendations at 4° C. for 30 min in the dark, washed twice with PBS and kept on ice for FACS analysis.
[0096]Briefly, 96-well plates were coated with anti human IgG antibody (Sigma: I 1886) diluted in 0.05 M Carbonate-Bicarbonate buffer (pH 9.7), and incubated at 4° C. for 16 h. Plates were blocked with blocking buffer (10 mM Tris, 0.15 M NaCl, 1% skim milk, pH 8.0) at 37° C. for 30 min. Culture supernatants were loaded on the wells and incubated at 37° C. for 2 h. Horseradish peroxidase conjugated anti-human IgG-F(c) antibody (Abeam: ab7499), diluted in dilution buffer (10 mM Tris, 0.15 M NaCl, 0.05% Tween 20, pH 8.0) according to the manufacturer's recommendations and incubated at 37° C. for 1 h. The reactions were detected using the substrate (1-Step® ultra TMB-ELISA, Pierce, Cat. 34028) and plates were read on microplate reader (Bio-Rad).
[0097]FIG. 8 presents a histogram profile of Herceptin (CHO.sup./+GFP/-dhfr) cells sorted at different fluorescent intensities using FACS. Panel A shows the GFP fluorescence in a pool of Herceptin CHO.sup./+GFP/-dhfr cells. The cells were divided into several subpopulations (R2, R3 and R4) based on the fluorescent intensity. Panels B-D show levels of GFP expression in each sub-population. Panel E shows the anti-herceptin antibody titles in the pool and each subpopulation. The data showed that cells with the lowest level of GFP fluorescence (Herceptin/R4/(CHO.sup./+GFP/-dhfr)) had the highest level of anti-herceptin titer.
[0098]FIG. 9 shows that the GFP expression level in CHO.sup./+GFP/-dhfr cells was reduced upon transfection with DNA constructs containing a target protein (Herceptin) and shRNAiGFP. The FACS profiles indicate that the GFP expression level was further reduced after two rounds of MTX challenge (panels C and D). These results demonstrate that the level of target gene amplification correlated to GFP expression levels and the strength of gene amplification.
[0099]FIG. 10 shows a representative FACS analysis of Herceptin/CHO.sup./+GFP/-dhfr cells stained with PE-conjugated mouse anti-human IgG(Fc). The result demonstrated that the antibody-producing cells (i.e., cells with higher PE staining) exhibited low levels of GFP expression.
[0100]FIG. 11A shows a histogram profile for Herceptin (CHO/+GFP/-dhfr) cells. Horizontal bars and shaded regions indicate the low-fluorescence and high-fluorescence cell populations used for the single cell analysis. FIG. 11B shows that 8 out of 18 low-fluorescent cell clones yielded ELISA values greater than 0.3, while only 2 out of 18 high-fluorescent cells clones yielded ELISA values grater than 0.3. These results demonstrated that the low-fluorescent cell population contains a higher frequency of high-yield antibody producing cells and confirmed the reversal GFP-based screen strategy.
[0101]FIG. 12 shows that GFP expression in CHO+GFP/-dhfr cells is reduced by the transfection of the DNA construct encoding Herceptin and shRNAGFP.
[0102]The use of the shRNAmireGFP coupled with flow cytometry substantially improves the accuracy and efficiency of cell line development at two crucial points. First, for early stage clone screening, the FACS method is a better predictor of clone productivity than the analysis of conditioned media for therapeutic protein titer. Second, clones with unstable transgene expression are easily identified by an observed increase in fluorescence during amplification stage. Thus, the present method provides the novel benefits of accurate 96-well clone screening to identify good candidates for further development and elimination of unstable clones at an earlier stage in the development process than traditional methods.
[0103]These results demonstrate that the DNA constructs and screening methods of the present invention reliably yield high-expression clones homogenous protein preparations. Since the isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is not performed by screening multiple individual clones with limiting dilution techniques, the procedure is less labor-intensive and may significantly reduce the time required to generate clones for bioproduction. The procedure requires no additional reagent for clone selection and offers additional benefits to monitor genome stability. The expression level of the target gene is also more correlative to the strength of amplification gene. The bicistronic design allows synchronously expressions of two foreign genes in the same chromosome. The inhibitive approach enhance the intensity of the target gene amplification by reducing the intensity of the reporter gene amplification. The use of ARE increases the expression of the foreign recombinant protein by eliminating the difference caused by the inhibition of inserting the foreign gene into the different chromosome areas.
Sequence CWU
29197DNAartificialsynthetic oligo 1tgctgttgac agtgagcgag cacaagctgg agtacaacta tagtgaagcc acagatgtat 60agttgtactc cagcttgtgc ctgcctactg cctcgga 97240DNAartificialsynthetic oligo 2cagaaggacc ggtaaggtat attgctgttg acagtgagcg 40337DNAartificialsynthetic oligo 3ctaaagtagc cccttaagct ttccgaggca gtaggca 374106DNAartificialsynthetic oligo 4ttgctctgag ccagcccacc agtttggaat gactcctttt tatgacttga attttcaagt 60ataaagtcta gtgctaaatt taatttgaac aactgtatag tttttg 1065106DNAartificialsynthetic oligo 5ttagaaatcc tcacacacaa caagttttca tttcacttct aattctgaaa aaaacactgc 60caccattttt tttccttccc ccaaccagca aaaactatac agttgt 1066106DNAartificialsynthetic oligo 6gtgtgtgagg atttctaatg acatgtggtg gttgcatact gagtgaagcc ggtgagcatt 60ctgccatgtc accccctcgt gctcagtaat gtactttaca gaaatc 1067106DNAartificialsynthetic oligo 7tggcagaaat gcaggctgag tgagactacc cagagaagag accggatata cacaagaagc 60atggtttata tcaatctttt gagtttagga tttctgtaaa gtacat 106825DNAartificialsynthetic oligo 8ttgctctgag ccagcccacc agttt 25930DNAartificialsynthetic oligo 9gttattaatt ggcagaaatg caggctgagt 301030DNAartificialsynthetic oligo 10cccacatgtt ggcagaaatg caggctgagt 301130DNAartificialsynthetic oligo 11ggactagttg gcagaaatgc aggctgagtg 3012370DNAMus musculus 12ttgctctgag ccagcccacc agtttggaat gactcctttt tatgacttga attttcaagt 60ataaagtcta gtgctaaatt taatttgaac aactgtatag tttttgctgg ttgggggaag 120gaaaaaaaat ggtggcagtg tttttttcag aattagaagt gaaatgaaaa cttgttgtgt 180gtgaggattt ctaatgacat gtggtggttg catactgagt gaagccggtg agcattctgc 240catgtcaccc cctcgtgctc agtaatgtac tttacagaaa tcctaaactc aaaagattga 300tataaaccat gcttcttgtg tatatccggt ctcttctctg ggtagtctca ctcagcctgc 360atttctgcca 370139729DNAartificialplasmid 13ttgctctgag ccagcccacc agtttggaat gactcctttt tatgacttga attttcaagt 60ataaagtcta gtgctaaatt taatttgaac aactgtatag tttttgctgg ttgggggaag 120gaaaaaaaat ggtggcagtg tttttttcag aattagaagt gaaatgaaaa cttgttgtgt 180gtgaggattt ctaatgacat gtggtggttg catactgagt gaagccggtg agcattctgc 240catgtcaccc cctcgtgctc agtaatgtac tttacagaaa tcctaaactc aaaagattga 300tataaaccat gcttcttgtg tatatccggt ctcttctctg ggtagtctca ctcagcctgc 360atttctgcca attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 420ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 480ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 540cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 600atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 660cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 720attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc 780ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg 840gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgtt 1020gccttcgccc cgtgccccgc tccgcgccgc ctcgcgccgc ccgccccggc tctgactgac 1080cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg 1140cttggtttaa tgacggctcg tttcttttct gtggctgcgt gaaagcctta aagggctccg 1200ggagggccct ttgtgcgggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg 1260ggagcgccgc gtgcggcccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg 1320gctttgtgcg ctccgcgtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc 1380gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag 1440ggggtgtggg cgcggcggtc gggctgtaac ccccccctgc aaccccctcc ccgagttgct 1500gagcacggcc cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg 1560ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg 1620gagggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg 1680agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc 1740caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg 1800gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg 1860cgccgccgtc cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt 1920cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc 1980tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt 2040attgtgctgt ctcatcattt tggcaaagaa tttaatacga ctcactatag ggagacccaa 2100gctggctagc gctaccggac tcagatctcg aggccggcaa ggccggatcc agacatgata 2160agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 2220tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 2280aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtggg gaggtttttt 2340aaagcaagta aaacctctac aaatgtggta tggctgatta tgatccggct gcctcgcgcg 2400tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 2460tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 2520gtgtcggggc gcagccatga ggggtaccat acattgaatc aatattggca attagccata 2580ttagtcattg gttatatagc ataaatcaat attggctatt ggccattgca tacgttgtat 2640ctatatcata atatgtacat ttatattggc tcatgtccaa tatgatttgc tctgagccag 2700cccaccagtt tggaatgact cctttttatg acttgaattt tcaagtataa agtctagtgc 2760taaatttaat ttgaacaact gtatagtttt tgctggttgg gggaaggaaa aaaaatggtg 2820gcagtgtttt tttcagaatt agaagtgaaa tgaaaacttg ttgtgtgtga ggatttctaa 2880tgacatgtgg tggttgcata ctgagtgaag ccggtgagca ttctgccatg tcaccccctc 2940gtgctcagta atgtacttta cagaaatcct aaactcaaaa gattgatata aaccatgctt 3000cttgtgtata tccggtctct tctctgggta gtctcactca gcctgcattt ctgccaacta 3060gtaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac 3120ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 3180aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 3240ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 3300gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 3360cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 3420cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat 3480tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggc 3540gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg 3600cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc 3660ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgttg ccttcgcccc 3720gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc 3780cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat 3840gacggctcgt ttcttttctg tggctgcgtg aaagccttaa agggctccgg gagggccctt 3900tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg 3960tgcggcccgc gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc 4020tccgcgtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg ggggggctgc 4080gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc 4140gcggcggtcg ggctgtaacc cccccctgca accccctccc cgagttgctg agcacggccc 4200ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg 4260gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg 4320ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca 4380ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc 4440ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg 4500cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc 4560ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg 4620gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca 4680tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc 4740tcatcatttt ggcaaagaat ttaatacgac tcactatagg gagacccaag ctggctagag 4800cttcgaattc tgcagtcgac agatccaccg gtgcccctct ccctcccccc cccctaacgt 4860tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac 4920catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag 4980cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa 5040ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag 5100gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga 5160tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag 5220agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc 5280ccattgtatg ggatctgatc tggggcctcg gtaacatgct ttacatgtgt ttagtcgagg 5340ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga 5400tgataatatg gccacaacca tggttcgacc attgaactgc atcgtcgccg tgtcccaaaa 5460tatggggatt ggcaagaacg gagacctacc ctggcctccg ctcaggaacg agttcaagta 5520cttccaaaga atgaccacaa cctcttcagt ggaaggtaaa cagaatctgg tgattatggg 5580taggaaaacc tggttctcca ttcctgagaa gaatcgacct ttaaaggaca gaattaatat 5640agttctcagt agagaactca aagaaccacc acgaggagct cattttcttg ccaaaagttt 5700ggatgatgcc ttaagactta ttgaacaacc ggaattggca agtaaagtag acatggtttg 5760gatagtcgga ggcagttctg tttaccagga agccatgaat caaccaggcc acctcagact 5820ctttgtgaca aggatcatgc aggaatttga aagtgacacg tttttcccag aaattgattt 5880ggggaaatat aaacttctcc cagaataccc aggcgtcctc tctgaggtcc aggaggaaaa 5940aggcatcaag tataagtttg aagtctacga gaagaaagac taagcggccg cgactctaga 6000tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 6060tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 6120cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 6180cactgcattc tagtttgctc tgagccagcc caccagtttg gaatgactcc tttttatgac 6240ttgaattttc aagtataaag tctagtgcta aatttaattt gaacaactgt atagtttttg 6300ctggttgggg gaaggaaaaa aaatggtggc agtgtttttt tcagaattag aagtgaaatg 6360aaaacttgtt gtgtgtgagg atttctaatg acatgtggtg gttgcatact gagtgaagcc 6420ggtgagcatt ctgccatgtc accccctcgt gctcagtaat gtactttaca gaaatcctaa 6480actcaaaaga ttgatataaa ccatgcttct tgtgtatatc cggtctcttc tctgggtagt 6540ctcactcagc ctgcatttct gccacttaag gcgtaaattg taagcgttaa tattttgtta 6600aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc 6660aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg 6720aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 6780cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc 6840cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 6900ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg 6960gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta 7020cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 7080ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 7140taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt 7200tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 7260attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 7320gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 7380taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 7440cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 7500gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt 7560gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat 7620gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 7680gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac 7740gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 7800gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 7860ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 7920ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag 7980cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat 8040caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag 8100gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 8160ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 8220ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg 8280ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 8340ttcttctgag cgggacaccg gtaaggtata ttgctgttga cagtgagcga gcacaagctg 8400gagtacaact atagtgaagc cacagatgta tagttgtact ccagcttgtg cctgcctact 8460gcctcggaaa gcttaagggg ctactttaga tcacgagatt tcgattccac cgccgccttc 8520tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 8580ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag 8640gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac 8700ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat 8760accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac 8820cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc 8880atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 8940aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 9000tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 9060tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 9120ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 9180ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 9240gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 9300aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 9360ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 9420agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 9480aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 9540aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 9600ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 9660cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 9720tctgtggat 972914124PRTHomo sapiens 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 12015372DNAHomo sapiens 15gaggtgcagc tcgtggagag tggtggcggg ttggtccagc caggcgggtc tctgcgattg 60agctgtgctg cctctggatt taacatcaaa gacacgtaca tccattgggt gagacaggcc 120cccggcaagg gccttgaatg ggtagctaga atctatccca ctaacggcta caccagatac 180gctgatagcg ttaaaggaag gtttactatt tctgccgaca cctccaagaa taccgcatat 240ctacagatga actccctgcg cgctgaggac accgctgtgt attactgctc acgttggggg 300ggagacggat tctacgcaat ggactactgg ggccagggta ccttagtgac agttagcagt 360gcctctacta ag 37216108DNAartificialsynthetic oligo 16gaggtgcagc tcgtggagag tggtggcggg ttggtccagc caggcgggtc tctgcgattg 60agctgtgctg cctctggatt taacatcaaa gacacgtaca tccattgg 10817105DNAartificialsynthetic oligo 17tttaacgcta tcagcgtatc tggtgtagcc gttagtggga tagattctag ctacccattc 60aaggcccttg ccgggggcct gtctcaccca atggatgtac gtgtc 10518105DNAartificialsynthetic oligo 18tacgctgata gcgttaaagg aaggtttact atttctgccg acacctccaa gaataccgca 60tatctacaga tgaactccct gcgcgctgag gacaccgctg tgtat 10519107DNAartificialsynthetic oligo 19cttagtagag cactgctaac tgtcactaag gtaccctggc cccagtagtc cattgcgtag 60aatccgtctc ccccccaacg tgagcagtaa tacacagcgg tgtcctc 1072030DNAartificialsynthetic oligo 20gccaagcttg aggtgcagct cgtggagagt 302130DNAartificialsynthetic oligo 21agggggccct tagtagaggc actgctaact 3022107PRTHomo sapiens 22Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10523321DNAHomo sapiens 23gatatacaga tgacacagtc tccgtcaagt ctgagcgcaa gcgtgggcga ccgggtaaca 60attacctgta gagccagcca ggacgtaaat acagccgtgg cctggtatca gcaaaaacct 120ggtaaagctc ctaagcttct gatctactct gcctcgttcc tttatagcgg ggtgccaagc 180cgcttctccg gatctaggtc tggaacagac tttactctga ccatttccag tctccagccc 240gaagactttg ctacctacta ttgccagcaa cattacacga ctccacccac atttgggcag 300ggcaccaagg tcgagatcaa g 3212493DNAartificialsynthetic oligo 24gatatacaga tgacacagtc tccgtcaagt ctgagcgcaa gcgtgggcga ccgggtaaca 60attacctgta gagccagcca ggacgtaaat aca 932595DNAartificialsynthetic oligo 25ccgctataaa ggaacgaggc agagtagatc agaagcttag gagctttacc aggtttttgc 60tgataccagg ccacggctgt atttacgtcc tggct 952694DNAartificialsynthetic oligo 26ctcgttcctt tatagcgggg tgccaagccg cttctccgga tctaggtctg gaacagactt 60tactctgacc atttccagtc tccagcccga agac 942793DNAartificialsynthetic oligo
27cttgatctcg accttggtgc cctgcccaaa tgtgggtgga gtcgtgtaat gttgctggca 60atagtaggta gcaaagtctt cgggctggag act 932830DNAartificialsynthetic oligo 28gccaagcttg atatacagat gacacagtct 302930DNAartificialsynthetic oligo 29cgcggattcc ttgatctcga ccttggtgcc 30
Patent applications by Min-Pey Ding, Taipei TW
Patent applications by Wei-Kuang Liu, Taipei City TW
Patent applications by ScinoPharm Taiwan Ltd.
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text:
Filipino Chicken Pastel
|Chorizo sausage/Chinese sausage
|Boneless chicken breast
|2 Pound, cubed
|Chorizo/Chinese sausage
|Beef franks/Vienna sausage, hotdogs
|1 , cubed
|1 , cubed
|2 Tablespoon
|Chicken stock
|1 Cup (16 tbs)
|Green olives
|Soy sauce
|3 Tablespoon
|Lemon/5 pieces calamansi
|White mushrooms/Button mushroom
|1 Cup (16 tbs), sliced
|1 Cup (16 tbs)
|1 Teaspoon
|1 Teaspoon
|3 Cup (48 tbs)
1. Marinade the chicken in lemon and soy sauce mixture for a minimum of 10 minutes
2. In a pot, place 3 cups of water and bring to a boil
3. Add the salt and pepper then put-in the chicken
4. Let the chicken cook until liquid completely evaporates. Set aside
5. On the same pan, apply heat and place the butter
6. Put-in the tenderized chicken and fry until the color turns golden brown
7. Add the Chorizo (or Chinese sausage) and beef franks (or Vienna sausage, hotdogs) and simmer for 5 minutes
8. Pour the chicken stock and bring to a boil
9. Put-in the carrots, potatoes, mushrooms, and green olives. Mix and simmer for 5 minutes
10. Thicken the sauce by adding flour and butter mixture (please see procedures below on how to make this)
11. Put the chicken in an oven-safe container and cover the top with dough (The dough will eventually be the pie crust after baking.)
12. Place in the oven and bake in 400 degrees Fahrenheit for 40 minutes
How to make the flour and butter mixture:
1. Heat a small pan and put-in 3 tbsp of butter
2. Add the flour and mix well
3. Pour ½ to 1 cup of water and mix thoroughly
How to make the Pie Crust:
1. In a large container, combine the following: 3 cups flour, ½ cup butter, ¼ tsp salt, and 3 tsp water
2. Mix the combined ingredients thoroughly (this will become the dough)
3. Knead the dough. Make sure that everything is properly distributed
4. Flatten the dough by using a rolling pin
5. Fit the dough on top of the container and trim according to the container size
6. Using a fork, shape the edge of the dough by pressing
7. Cover the edges with aluminum foil to prevent over cooking
8. Bake in 400 degrees Fahrenheit for 40 minutes
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 4492 Calories from Fat 2952
% Daily Value*
Total Fat 329 g506.5%
Saturated Fat 173 g864.8%
Trans Fat 6 g
Cholesterol 1373.3 mg457.8%
Sodium 9124.4 mg380.2%
Total Carbohydrates 104 g34.8%
Dietary Fiber 12.3 g49.2%
Sugars 15.8 g
Protein 280 g559.2%
Vitamin A 330.8% Vitamin C 171.1%
Calcium 31.5% Iron 76%
*Based on a 2000 Calorie diet

subset: commoncrawl


text:
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text: Pensión La Palma is located just 329 feet from Larios Street and the Alameda Boulevard, in Málaga's historic centre. It offers a charming sun terrace and functional rooms with free Wi-Fi and LCD TVs. Most rooms at Pensión La Palma have a balcony, and all rooms offer a fan and a desk. Some rooms have a private bathroom, while others have access to shared bathroom facilities. A kettle and fridge are available in the lobby. Staff at the guest house's 24-hour reception are happy to store your luggage or provide information about the city. The streets around Pensión La Palma are lined with shops, bars and restaurants. Málaga Port is just 1,149 feet away, while La Malagueta Beach is a 20-minute stroll away.


text:
Where you can catch Oscar-nominated films in Milwaukee
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But there's more here than initially meets the eye, as we learn when the woman sharing Nick's dorm-room bed begins asking about the web of scars along his waistline. Does Nick tell Molly the truth about these now-healed wounds? Or should he do as he's always done and make "stuff" — Nick uses a different word — up? »Read Full Article(1)
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text:
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Brace for Lions action: The Breakdown
Harbour upset Otago to earn NPC promotion
Larkham wants Cheika's job
Koroibete: Cheika never promised me anything
Hansen responds to Cheika rant
Cheika says NZ lack respect
Australia's controversial no-try
Black Ferns too good for Wallaroos
Brace for Lions action: The Breakdown
The Waratahs have assembled an impressive back line against the Lions in what should be a cracker of a game this weekend.
It is a major concession to the provinces and to the Lions, who have spent the past two weeks remarking on the relative weakness of their opponents when stripped of their Test players.
The decision will also give Wallabies coach Robbie Deans an inside line into the tourists' playing style when Dennis, Horne, Kimlin and Sio go back into camp after the completion of their matches.
And it turns the Waratahs match on Saturday into a genuine competition, allowing NSW coach Michael Cheika to field something near his complete starting back line, with just Adam Ashley-Cooper and Israel Folau now missing.
Regular Waratahs five-eighth Bernard Foley was yesterday released from Australia's Sevens World Cup camp, along with second-choice halfback Matt Lucas.
The return of Dennis against the Lions will give the Waratahs some much needed leadership, with flanker Pat McCutcheon still in doubt with a knee knock.
The Waratahs name their side to take on the Lions on Friday morning. The Lions are expected to name their team today.


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Karanvir Bohra, Teejay Sindhu became a proud parents of twin daughters. Teejay delivered twin daughters in Canada and the mother and the daughters are said to be doing fine. The couple welcomed baby after tens years of blissful marriage. It definitely called for a double celebration.
Prem and Mukti scene 28
Mukti and Prem talk in Prem's office. No infringement intended.
Karanvir Bohra
Television actors Karanvir Bohra and wife Teejay Sidhu are expecting their first child after almost 10 years of their marriage. The child is due in November, the same month when the couple will celebrate their 10th wedding anniversary. Teejay, who is currently with her family in Vancouver,
Television actor Karanvir Bohra and his actress wife Teejay Sidhu are expecting their first child in November. The two have been are married for almost 10 years now and the to-be-parents are very much thrilled about this new phase of their life. An excited Karanvir recently shared a pictur
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text: Guest accommodation comprises 36 rooms. Services and facilities at the hotel include a restaurant and a bar. Those arriving in their own vehicles can leave them in the car park of the hotel. The accommodation features rooms with air conditioning and a bathroom. A balcony is included as standard in most rooms, offering additional space for relaxation. Guests can enjoy a good nights' sleep on the double bed. All rooms feature a desk and a TV. The accommodation units include a bathroom with a shower. A hairdryer can also be found in each of the bathrooms. The establishment offers non-smoking rooms. Copyright GIATA 2004 - 2016.


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The Orlando Magic have been involved in several trade scenarios that mainly concern "you know who."
They are winning now and can beat most teams in this league. Perhaps they need a piece or two to put them over the top.
I'd like to break down a few of the trade assets that the Magic have to offer. They are currently first place in their division, but they may be inclined to bring in a piece or two to keep a certain superstar in The City Beautiful.


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HourlyHours to be determined
Start DateFebruary 10, 2013
Hi I saw your profile. I need someone to do SEO for my website pjpportraits.co.uk for the following keywords
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About the Client
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Marcus Nilsson
Oltranti doesn't consider himself a baker, but he should. We all agree that his biscotti are among the best weve had. Starting with a lot of almonds in a sticky dough, his recipe produces crunchy cookies that taste even better a day or two later—if they last that long.
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This recipe is perfect in everyway. I would prefer weight measurements, I use concentrated almond emulsion instead of almond extract and I didn't hither toasting almonds, though I'm sure it would be better. I just love this recipe and would suggest doubling the batch and storing well.
I've been trying to find the perfect biscotti recipe for quite awhile. I'm not sure this is it. It is definitely easy to work with, but it doesn't seem to have much flavor. I felt like I was eating flour. However, my husband really liked them especially when I put chocolate over the ends. I think I will try them again using differnt flavor. I made them exactly as the recipe indicated. I will say they held up well when dunking in coffee and that's a big plus.
This recipe is as wonderful as everyone says it is. It's in my "saved" folder now! Only crumbs left, unfortunately; have to get baking!
A Keeper!! I made these exactly 100% as was directed in the recipe & they are great! I wouldn't change a thing or do anything different. They came out with the texture & flavor that a good biscotti should have. They are a nice light desert & go well with coffee or tea. People who don't like overly sweet deserts will be able to really appreciate these. My Father is one of them. These were simple to make & didn't really take very long. The dough is very easy to handle as long as you moisten your hands as suggested, the dough really doesn't need much handling & this recipe was a pleasure to work with.
I've made biscotti recipes that are better and easier to handle. Despite chilling the dough for over an hour, it was far stickier than others I've made. I added chopped bittersweet chocolate and dried cherries, but there just isn't much flavor. Hopefully, it will improve by tomorrow when I serve it.
This is a delicious and easy recipe. Just a couple changes; I used Kahlua (I didn't have Brandy) and I used slivered almond, which, I think makes it easier when cutting. I did not need to cook them 25 minutes after cutting but I did turn them over half way thru the toasting process. These got rave reviews and I am making them again this weekend.
Lia_Branden, is it possible that you are using old baking powder? Baking soda lasts forever but baking powder(maybe because of it's starch content) is more sensitive to moisture. You can check out this David Lebovitz article on how to check to make sure your baking powder is still active. http://www.davidlebovitz.com/2011/11/how-to-tell-if-baking-powder-is-still-good/
Soooo this was my go-to recipe for months and months and I have used this recipe countless times. Something strange happened, my last 3 attempts at making this, failed miserably. Dough doesn't rise, it's very wet even after being chilled in fridge for 30 mins. Dough doesn't rise in the oven. It's the strangest thing. Anyone has experienced this? Does anyone know why this happens? HELP!! Thank you.
Delectable and easy. Added orange zest and used an apple brandy. Drizzled with a bit of melted dark chocolate, more for the look than anything else. Better than any you can buy in a cafe and was perfect to send in care packages.
The place we buy our biscotti from is 26 miles away and it is winter in MN. Today, I searched through many cooking sites and decided to try this recipe to avoid a trip in -20 degree wind chill temps. I made a double batch, leaving the almonds till later. One includes almonds, dried cherries and Kirsch liquor instead of brandy--delicious already. The other has chocolate chips and my own dried wild blueberries--equally sublime. I will definitely be using this as a base recipe for future experimentation.


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Dr. Patricia Abinoja specializes in pediatric medicine in Oak Park, Michigan.
Dr. Patricia Abinoja has been compared 28 times. Details of Dr. Abinoja's experience as an MD and her hospital affiliation are available on your free doctor report.
Primary Location for Dr. Abinoja
21040 Greenfield Rd
Oak Park, MI 48237
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Board Certification, Specialty and Expertise
Dr. Patricia Abinoja has been identified as specializing in Pediatric Medicine.
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Dr. Patricia Abinoja accepts the following plans at 21040 Greenfield Rd, Oak Park, MI
- Aetna
- Humana
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Dr. Abinoja is affiliated with 1 hospital:
30671 Stephenson Hwy
Madison Heights, Michigan
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