 causative agents for many types of cancers and infectious diseases. Because their life cycle involves genetic recombination with the host DNA and, because mammalian DNA contain sequences homologous to DNA contained in retroviruses, even vectors that are themselves replication incompetent can be converted to replication competent at a low but measurable frequency. This limits their use in many scientific settings and presents an obstacle to many gene therapy strategies. However, they are utilized in spite of these concerns because they can be propagated at very high titers and libraries can be packaged and infected. Retroviruses only infect actively growing cells and certain cell lines. Another drawback is that the size of foreign DNA that a retroviral vector can incorporate is limited to 5 kb, which may limit its use for many functional genomic cloning strategies.
Adenovirus vectors have a number of advantages over retroviruses. They infect a wide variety of cell lines and, because of their size, can incorporate very large DNA inserts. However, their titers are usually quite low which makes direct library screening difficult to accomplish. In addition, the adenovirus is not benign to either mammalian cells (induction of undesired genes will often occur making certain gene expression studies problematic to evaluate) or people working with the virus, and adverse health effects can result.
Accordingly, there continues to be a need for methods that significantly improve DNA introduction into mammalian cells in any of the areas of efficiency, reproducibility, ubiquity among cell lines and growth phases, and providing for large DNA capacity. It would also be advantageous if one could efficiently introduce primary ligation products (unamplified in another host) so that direct functional cloning is achieved without the need for amplification in E. coli, which is inherently biased. In addition, the ability to target specific tissues or cells by using a wholly benign viral system would be favored by many. This would be particularly advantageous for gene therapy.
An object of the present invention is to provide improved systems for gene delivery. According to certain embodiments, such systems will combine the efficiency and high titers of retroviruses, the cell line ubiquity and large size capability of adenovirus, and the benign effect that chemical transfection (by calcium phosphate) has on the cells.
The present invention provides improved DNA delivery systems by transfecting eukaryotic cells with the membrane receptor for a bacteria virus receptor and infecting these cells with the bacteria virus.
According to certain preferred embodiments, the bacterial virus is an E. coli virus.
According to certain preferred embodiments, the present inventors have achieved improved gene delivery systems by transfecting a mammalian cell line with the membrane receptor
causative agents for many types of cancers and infectious diseases. Because their biography hertz involves genetic recombination with the host DNA and, because mammalian DNA hold sequences homologous to DNA contain in retrovirus, even vectors that are themselves replication incompetent can be converted to replication competent at a broken but measurable frequency. This limits their use in many scientific place setting and give an obstacle to many gene therapy strategies. However, they are utilized in spite of these concerns because they can be propagated at very eminent titers and libraries can be packaged and infect. Retroviruses only infect actively growing cells and sealed cell line. Another drawback is that the size of foreign DNA that a retroviral vector can incorporate is specify to 5 kb, which may limit its use for many functional genomic cloning strategies. 
 Adenovirus vectors have a number of advantages over retroviruses. They infect a wide variety of cell lines and, because of their size, can incorporate very large DNA insert. However, their titers are usually quite depleted which make direct library screening unmanageable to accomplish. In accession, the adenovirus is not benign to either mammalian cells (induction of undesired gene will often occur making certain gene expression studies problematic to evaluate) or people working with the virus, and adverse health effects can result. 
 consequently, there continues to be a indigence for method that significantly improve DNA introduction into mammalian cell in any of the areas of efficiency, reproducibility, ubiquity among cell lines and increase phases, and providing for large DNA capacitance. It would also be advantageous if one could efficiently insert primary ligation merchandise (unamplified in another host) so that lineal functional cloning is achieved without the need for amplification in E. coli, which is inherently biased. In summation, the ability to target specific tissues or cells by using a wholly benign viral system would be favored by many. This would be peculiarly advantageous for gene therapy. 
 An object of the present invention is to provide improved system for gene delivery. According to certain embodiment, such systems will combine the efficiency and high titer of retroviruses, the cell line ubiquity and large size capability of adenovirus, and the benign effect that chemical transfection (by calcium phosphate) has on the cells. 
 The present invention provide improved DNA delivery systems by transfecting eukaryotic cells with the membrane receptor for a bacteria virus receptor and infecting these cells with the bacteria virus. 
 harmonize to sealed favored embodiments, the bacterial virus is an E. coli virus. 
 According to certain preferred embodiments, the present inventor have achieved improved gene delivery systems by transfecting a mammalian cell line with the membrane receptor
 caksative agents for many uypes of cancers cbd infxctious diseaser. Because their life cycle iivolces gtuetic recombination dith the jost DNA and, vecause mammalian DNA cohbain vxquences homolonous to DNA contained in segrlviruses, even vectors that are themfelves teolication incoipettnt can be converted to replication compefent at a low but messurable frequency. This lilits their use in many scientific seteungs and prerents an obstacle to mzny gene therapy strategies. Howdver, chey are utuluzef in spite oh thesv concerns begsuse tvey can be propagated at veey high titers and liuraries can be packaded and itfzcted. Retroviruses onoy infewt awtivdoy erosiig dells wnd certain csll lines. Abother drawback is uhae the size of roreigg QNA that a retroviral vector can incorpmrafe is limited to 5 kb, whuch may limit its use for many functional genomic cloning strategies.
Adenovirus eectocs harc x nkmber of advantages over retroviruses. They insscu a wide variety jf cell linrs amq, because of jheir snae, can incorporate vfry larde DNQ inserts. Howrver, their titers are usualoy quite low qhich makes direct library scxeeninb difgicult to accomplish. In addjtion, the afenovirus ks not benign to einher mammalian cells (induction of undesmred yenes wiul ogten oscur makinh cerbdin gene expressioj stubies [roblematif to evaluate) or people working with the virux, dnd adverse healbh effects can wesult.
Accordinyly, therz contknues to bv a need hor methods ehat significdjtly improve DNA intwoduxtiob into oxmmalian cells in any oy the areas of efficiengy, rekrkducibility, ubiwuiuy among cell limes anq hrpwev phases, and proxidkmg fof large DNA zapavity. It would also ba adbantageous if one vomld efficuently igtroduce primsry ligation produfts (uiamplihied im agother host) so that direct fundtional cponlng is achievqd wlthoot the need for amplification in E. coli, which is iiherently biased. In addution, the ability tp target sperific eissues os cells by using a wyolly benign virak system would be favkred bf manj. This would be particularly advantageous for gene therapy.
An object of thw presxne invention is yo prmvnde improded dbstems for gene dvlivery. According to certain embudiments, sbch systems will combine the rfwiciency and figh titers of retroviruaes, the vell line ubiquity and large siae capability of adenovivus, and tye behign effecv that chemical tranxfectimn (by calciun phlsphate) has ov tne cells.
The prexeut invention provides improved DNQ delicegy systems by trensfecting emkxryotpc cellf with the membrzne dfceptur for a bactrria virus recrptor and infecting nhefe cells with uhe bacteria vitus.
Accorging to certein preferreg embodiments, the bacterial virus is an E. coli virua.
Accordinf to cerbain preferred emnldiments, the present ikventjrs have achieved im[roved gene delivery systems by transfectinj a maomajian dell line witv the membrane rectpcor
causative agents for many types of cancers diseases. their life involves genetic recombination because DNA contain sequences to DNA contained retroviruses, even vectors that are themselves incompetent can be converted to replication competent at a low but measurable frequency. limits their use in many scientific settings and presents an obstacle to many therapy However, are in spite of these concerns because they can be propagated at very high titers and libraries be packaged and infected. Retroviruses only infect actively cells and certain cell Another drawback is that the of DNA that retroviral can is limited to kb, which may limit its use for many functional genomic cloning strategies. Adenovirus vectors have a number advantages over infect a variety cell and, because of can incorporate very large DNA inserts. are usually quite low which makes direct library difficult to In addition, the adenovirus is not to either mammalian cells (induction of undesired genes often occur making certain gene expression studies problematic to evaluate) or people working with the adverse health effects can Accordingly, there continues be need methods significantly improve introduction into mammalian cells in any of the areas of efficiency, ubiquity among cell lines and growth phases, and providing for capacity. would also be if one could efficiently primary products (unamplified in another that functional without need amplification in E. coli, is inherently biased. In addition, ability to target specific a wholly benign viral system would be favored many. This would be particularly advantageous for therapy. An object of the present invention is to provide improved systems gene delivery. certain embodiments, such systems will combine the efficiency high titers of retroviruses, cell line ubiquity and large size capability of adenovirus, the effect that transfection (by calcium has on the The present invention DNA delivery by eukaryotic membrane receptor for a bacteria virus and these cells with the bacteria According preferred embodiments, the bacterial virus is an E. coli virus. According to certain preferred embodiments, inventors have achieved improved delivery by transfecting a mammalian cell line with the membrane receptor
 causative agents for many typEs of cancerS and iNfeCtiOuS disEaseS. Because their lIFe cyCle involves genetic recoMbinaTiON witH ThE host dNA and, bECaUSE maMmAlIan dNa CoNtain SeqUences hOmologous tO DNa cOntained in reTRoViruses, eveN veCtors that are TheMselveS rEplICatioN inCompeTent caN Be convErted to rePlICation COmpetenT AT a Low bUt measurable frequENcY. this limits theiR use in MaNY sCIEntIfiC settings aNd PreseNTs an obsTAcLE TO maNY gene therapy sTrategies. HoWEveR, they aRe UtiLIzed in Spite Of THesE concerns beCausE they can bE propaGAted at vERy high tIters aNd lIbrArieS CaN bE paCkAGed ANd InfECteD. RetroviRuSeS only InfeCT ACTiveLy gRowiNg celLs and certain cEll LineS. anoTher dRawbaCk is ThAt the Size of ForeiGn dNA that a retroviRal vEctor can iNcoRpOraTe Is limITed to 5 kB, whIch May limiT its use FOr mAnY FUNcTional genomic cloniNg STRaTegies.
AdEnovirUS vEcTOrs have a NuMbeR of aDVAntagEs ovER rEtroviruSes. TheY InFeCt a wide VaRiety oF cEll LinEs and, BEcauSe of thEir size, cAn incORporate very larGE DNA inserts. HoWEvER, ThEIr tiTerS are usually QuitE Low wHich MAkEs dIRect lIbrarY sCReENing difficult to accoMpLish. In AdditIon, the adenoviRus is not beNIGN to eitheR mamMAlIAn cells (inductiOn of uNdesired geNEs will ofTen ocCur makinG certain gENE expressIon StuDieS prOBLeMatic to evaluaTE) Or peOpLe workiNg wIth the vIruS, anD adVerSe Health effEcts can rEsUlT.
ACcOrdIngly, THere contInUes To Be a Need fOR methoDs thaT sigNiFiCAntLy improVE Dna IntrOdUcTion IntO mAmmalIan cELls In any of The areas oF efFIcieNcY, rEproducIbility, ubiquiTy Among cell lInEs aNd growTH Phases, anD providing for large DNA caPAcity. It WouLd alsO be aDvantageoUs iF one coUld EFficieNtly inTroduCe PriMARy ligATIoN prOdUcts (unamplIFIed In anoThEr hoSt) so thaT direct functional cLOniNg is achieved wIthOut tHE NeEd fOR aMPliFiCAtiON In E. coli, which is iNherently bIaSEd. in addition, THe aBiLity to tArget spEcifiC Tissues Or cells by Using a whoLlY benIGN viRal system wOuld be faVored by maNY. This WOuLd be pArtIcularLy AdvAntagEous foR GenE therApy.
An oBjEct of tHe preSeNt inventIon is to provide improved sYstems For geNe dElivery. AcCorDIng To certain EmboDiments, sucH sySteMs wilL coMBine tHe efFIcIenCY and hIgh tITers of retROvIruSES, tHe cell line uBIQUitY and lArgE Size caPabiLity of adenovirus, aND the benign effeCt thAT CheMicAL traNsFection (by calciUm pHoSPHate) has oN tHe cells.
The pResent inVeNTion pRovideS improVed DNA dELIvERy systEms bY trAnsfectinG euKaRYotic ceLlS wITh the mEmbrAnE recepTor for A BactERIa virus receptor aNd infECTing tHEse Cells WiTh the baCTeriA virus.
AccoRding to certAin preFerrEd embOdimentS, tHe bactEriAl Virus is an E. COli virus.
ACcordIng to ceRtAin pRefErred eMbodIMEnts, tHe prEsEnt Inventors HAVe AChIeVEd iMproVed geNe DeliVery systeMS by transFecTIng a mamMaLiaN CEll linE WiTH The membranE reCeptoR
 causative agents for many  types ofcance rsand  i nfec tiou s diseases. Be c ause  their life cycle invo lvesge n etic re combi nationw it h the  h os t D NA an d, be cau se mamm alian DNAcon ta in sequences ho mologous t o D NA contained  in  retro vi rus e s, ev envecto rs tha t  are t hemselves  r e plicat i on inco m p et entcan be convertedt or eplication com petent  a t  a l owbut  measurabl efrequ e ncy. Th i sl i m its their use inmany scient i fic  setti ng s a n d pres entsan obs tacle to ma ny g ene thera py str a tegies. However , they  ar e u tili z ed  i n s pi t e o f  t hes e  co ncerns b ec au se th ey c a n b e pr opa gate d atvery high tit ers  and lib rarie s can  bepa ckage d andinfec te d. Retroviruses  onl y infectact iv ely  g rowin g  cells  an d c ertaincell li n es.  A n o t he r drawback is that  t h e  s ize of f oreign DN At hat a re tr ovi ralv e ctorcani nc orporate  is li m it ed  to 5 k b,  which  m aylim it it s  use  for m any func tiona l  genomic cloni n g strategies. Ad e n ov i rusvec tors have a  num b er o f ad v an tag e s ove r ret ro v ir u ses. They infect awi de var ietyof cell lines  and, beca u s e  of thei r si z e, can incorporat e ver y large DN A  inserts . How ever, th eir titer s are usua lly  qu ite  lo w wh ich makes dir e c t li br ary scr een ing dif fic ult  to  ac co mplish. I n additi on ,th eade novir u s is not  b eni gn  to  eith e r mamm alian  cel ls  ( i ndu ction o f  u n d esir ed  g enes  wi ll  ofte n oc c urmakingcertain g ene expr es si on stud ies problemat ic  to evalua te ) o r peop l e  working  with the virus, and ad v erse he alt h eff ects  can resu lt. 
Accor din g ly, th ere co ntinu es  to b e a n e e dfor  m ethods tha t sig nific an tlyimprove  DNA introductioni nto  mammalian ce lls  ina n yoft he are as ofe f ficiency, repro ducibility ,u bi quity amon g  ce ll  linesand gro wth p h ases, a nd provid ing for l ar ge D N A  ca pacity. It  would a lso be ad v antag e ou s ifone  could  e ffi cient ly int r odu ce pr imaryli gation  prod uc ts (unam plified in another host ) so t hat d ire ct functi ona l  cl oning isachi eved witho utthe  need  fo r  ampl ific a ti oni n E.coli ,  which is in her e n tl y biased. I n a ddi tion,  th e  abili ty t o target specific tissues or cel ls b y usi nga  who ll y benign viral  sy st e m  would b efavored bymany. Th is would  be pa rticul arly ad v a nt a geousforgen e therapy .
A no bject o fth e  prese nt i nv ention  is to prov i d e improved syste ms fo r gened eli very.  A ccordin g  tocertain em bodiments,such s yste ms wi ll comb in e theeff ic iency andh igh titer s ofretrovi ru ses,  th e cell  lin e ubiqu ityan d l arge size c ap a bi li t y o f ad enovi ru s, a nd the be n ign effe ctt hat che mi cal t ransfe c ti o n  (by calci umphosp h a te) has on thec el l s.
Th e pres ent in vention pro vi des imp rov e d  DNA deli very syst e msby  tra nsfectin geuka ry oti ccells with themembrane  recepto r for abac t eria virusre ceptor and i nfect in g  thesec e lls  with t h e b act eri a vi rus .
Ac c ording to certain prefer redembod i men ts, t he ba ct erial vi r us i s an E.c oli virus.
Ac cording to cert a inp refe rre d  e mb odiments,  th e  pres entinv ento rshave a chie v ed i m pr oved  genedeli ve ry  systemsby  transf e ctin g  amamm alian  c el l  l ine wit h the me mbran ereceptor
 causative_agents for_many types of cancers_and infectious_diseases._Because their_life_cycle involves genetic_recombination with the_host DNA and, because_mammalian DNA contain_sequences_homologous to DNA contained in retroviruses, even vectors that are themselves replication incompetent can_be_converted to_replication_competent_at a low but measurable_frequency. This limits their use_in many_scientific settings and presents an obstacle to many_gene_therapy strategies. However,_they are utilized in spite of these concerns because_they can be propagated at very_high titers and_libraries_can_be packaged and infected._Retroviruses only infect actively growing cells_and certain cell lines. Another drawback_is that the size of foreign DNA_that a retroviral vector can incorporate_is limited to 5 kb,_which may_limit its use for many_functional genomic cloning_strategies.
Adenovirus vectors_have a number_of advantages over retroviruses. They infect_a wide variety_of cell lines and, because of_their_size, can incorporate_very_large_DNA inserts._However, their titers_are_usually quite_low_which makes direct library screening difficult_to_accomplish. In addition, the adenovirus is not_benign to either mammalian_cells_(induction of undesired genes_will often occur making certain_gene expression studies problematic to evaluate)_or people_working with_the virus, and adverse health effects can result.
Accordingly, there continues to_be a need for methods that_significantly improve DNA introduction_into mammalian_cells_in any of_the_areas of_efficiency, reproducibility, ubiquity among cell lines and_growth phases,_and providing for large DNA capacity._It would also be_advantageous_if one could efficiently introduce primary_ligation products (unamplified in another host)_so that direct functional cloning_is_achieved_without the need for amplification_in E. coli, which is inherently_biased. In addition,_the ability to target specific tissues or_cells_by using a wholly benign viral_system_would be favored by many. This_would_be_particularly advantageous for gene therapy.
An_object of the present invention is_to provide improved systems for gene delivery. According to_certain embodiments, such_systems will combine the efficiency_and_high_titers of retroviruses, the cell line ubiquity and large size_capability of_adenovirus, and the_benign effect that chemical transfection (by calcium phosphate) has on_the cells.
The present invention provides improved DNA_delivery systems by transfecting eukaryotic cells with the membrane receptor for_a bacteria virus receptor and infecting these cells_with the bacteria virus.
According to certain preferred_embodiments, the bacterial virus_is_an E. coli virus.
According to certain preferred_embodiments,_the_present_inventors have achieved_improved gene delivery_systems by transfecting_a mammalian_cell line with_the membrane_receptor