WO1998059060A1 - Procedes de preformage de recombinaison homologue d'apres une modification d'acides nucleiques dans des cellules presentant une deficience de recombinaison, et utilisation des produits d'acides nucleiques modifies de celles-ci - Google Patents

Procedes de preformage de recombinaison homologue d'apres une modification d'acides nucleiques dans des cellules presentant une deficience de recombinaison, et utilisation des produits d'acides nucleiques modifies de celles-ci Download PDF

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WO1998059060A1
WO1998059060A1 PCT/US1998/012966 US9812966W WO9859060A1 WO 1998059060 A1 WO1998059060 A1 WO 1998059060A1 US 9812966 W US9812966 W US 9812966W WO 9859060 A1 WO9859060 A1 WO 9859060A1
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reca
gene
protein
host cell
tssv
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PCT/US1998/012966
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English (en)
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Nathaniel Heintz
Peter Model
Xiangdong W. Yang
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The Rockefeller University
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Priority claimed from US08/880,966 external-priority patent/US6143566A/en
Application filed by The Rockefeller University filed Critical The Rockefeller University
Priority to AU79848/98A priority Critical patent/AU730859B2/en
Priority to JP50494899A priority patent/JP2002515764A/ja
Priority to CA002294619A priority patent/CA2294619A1/fr
Priority to EP98930459A priority patent/EP0998574A1/fr
Publication of WO1998059060A1 publication Critical patent/WO1998059060A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/202—Pseudochromosomes, minichrosomosomes of bacteriophage origin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/204—Pseudochromosomes, minichrosomosomes of bacterial origin, e.g. BAC
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • This invention relates generally to methods of modifying genes with specificity in recombination deficient cells by transiently enabling homologous recombination in the cells. Included in the invention are conditional replication shuttle vectors which bestow transient recombination capabilities to an otherwise recombination deficient cell.
  • the independent origin based cloning vectors containing the modified genes and methods of using the independent origin based cloning vectors containing the modified genes are also included in the present invention.
  • the size of the genomic DNA that can be readily manipulated in vitro and introduced into the germline can be a critical determinant of the outcome of the functional analysis of a gene since elements that are important for high level, tissue specific and position-independent expression of the transgene may be located at a long distance from the gene itself [Dillon et al., Trends Genet. 9:134 (1993); Kennison, Trends Genet. 9:75 (1993); Wilson et al, Annu.Rev.Cell.Biol. 6:679 (1990)].
  • the use of such large genomic transgenes has several practical problems.
  • the size of the transgene is presently limited due to constraints on the sequence length that can be cloned and stably maintained in a conventional plasmid or a cosmid.
  • DNA sequences suspected of being nonessential are often omitted when designing the constructs to be transferred because of the size limitation.
  • in vitro manipulations of large DNAs oftentimes lead to mechanical shear [Peterson et al, TIG 13:61-66].
  • Yeast artificial chromosomes allow large genomic DNA to be modified and used for generating transgenic animals [Burke et al, Science 236:806; Peterson et al, Trends Genet. 13:61 (1997); Choi, et al., Nat. Genet., 4:117-223 (1993), Davies, et al, Biotechnology 11:911-914 (1993), Matsuura, et al, Hum. Mol Genet, 5:451-459 (1996), Peterson et al, Proc. Natl Acad. Sci., 93:6605-6609 (1996); and Schedl, et al, Cell, 86:71-82 (1996)].
  • YACs Yeast artificial chromosomes
  • YACs have certain advantages over these alternative large capacity cloning vectors [Burke et al, Science, 236:806-812 (1987)].
  • the maximum insert size is 35-30 kb for cosmids, and 100 kb for bacteriophage PI, both of which are much smaller than the maximal insert for a YAC.
  • YACs E. coli based cloning systems based on the E. coli fertility factor that have been developed to construct large genomic DNA insert libraries. They are bacterial artificial chromosomes (BACs) and P-l derived artificial chromosomes (PACs) [Mejia et al, Genome Res. 7:179-186 (1997); Shizuya et al, Proc. Natl. Acad. Sci. 89:8794-8797 (1992);Ioannou et al, Nat. Genet., 6:84-89 (1994); Hosoda et al, Nucleic Acids Res. 18:3863 (1990)]. BACs are based on the E.
  • coli fertility plasmid F factor
  • PACs are based on the bacteriophage PI.
  • the size of DNA fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules has been expanded by the advent of PACs and BACs.
  • These vectors propagate at a very low copy number (1-2 per cell) enabling genomic inserts up to 300 kb in size to be stably maintained in recombination deficient hosts (most clones in human genomic libraries fall within the 100-200kb size range).
  • the host cell is required to be recombination deficient to ensure that non-specific and potentially deleterious recombination events are kept to a very minimum.
  • Functional characte ⁇ zation of a gene of interest contained by a PAC or B AC clone generally entails transferring the DNA into a eukaryotic cell for transient or long-term expression.
  • a transfection reporter gene e.g , a gene encoding lacZ, together with a selectable marker, e.g., neo, can be inserted into a BAC [Mejia et al, Genome Res 7:179-186 (1997) Transfected cells can be then detected by stammg for X-Gal to verify DNA uptake.
  • Stably transformed cells are selected for by the antibiotic G418.
  • a cloning vector that has the capacity to contain greater than 100 kilobases of DNA, which can be readily manipulated and isolated, but still can be stably stored m libraries relatively free of rearranged clones.
  • methodology for generating such cloning vectors There is also a need to apply such vectors to improve current technologies such as gene targeting.
  • Gene targeting has been used in various systems, from yeast to mice, to make site specific mutations in the genome. Gene targeting is not only useful for studying function of proteins in vivo, but it is also useful for creating animal models for human diseases, and in gene therapy.
  • the technique involves the homologous recombination between DNA introduced into a cell and the endogenous chromosomal DNA of the cell. However, in the vertebrate system, the rate of homologous recombination is very low, as compared to random integration.
  • the only cell line that allows a relatively high homologous recombination rate and maintains the ability to populate the germline is the murine 129 embryonic stem cells (ES cells).
  • mice can be generated with a targeted mutation (Gene Targeting, a practical approach Ed. by A. Joyner, IRL Press: Oxford, New York, Tokyo).
  • Gene Targeting a practical approach Ed. by A. Joyner, IRL Press: Oxford, New York, Tokyo.
  • the rate of homologous recombination for some gene loci in ES cells is still extremely low ( ⁇ 1 %), the procedure is labor intensive, and the cost of generating targeted mutant mice is very expensive.
  • gene targeting in a germline is still not possible for other vertebrates.
  • the major limitation for gene targeting in vertebrate cells remain to be the low targeting frequency.
  • One critical factor affecting the targeting frequency is the total length of homology.
  • Deng and Capecchi (MCB, 12:3365-3371) have shown that gene targeting frequency is linearly-dependent on the logarithm of the total homology length over homology lengths of 2.8kb to 14.6kb. Since the curve did not plateau at the 14.6kb homology, it is likely that incorporating greater homology lengths into the targeting vector will further increase the homologous recombination rate.
  • gene targeting in TB has a very low rate, mainly due to the predominance of random integration over homologous recombination. It has been demonstrated that using a 40-50 kb linear targeting construct, a 6% targeting frequency could be obtained, whereas no targeting event was obtained at all with a smaller ( ⁇ lOkb) targeting construct [Balasubramanian et ah, J. of Bacteriology 178:273-279 (1996)]. Therefore, there is a need to construct large gene targeting constructs to allow efficient gene targeting in many biological systems.
  • the present invention provides a novel and efficient method of modifying independent origin based cloning vectors for in vitro and in vivo gene expression.
  • the present invention provides a method of selectively performing homologous recombination on a particular nucleotide sequence contained m a recombination deficient host cell, i.e., a cell that cannot independently support homologous recombination.
  • the method employs a recombination cassette which contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination deficient host cell is induced to support homologous recombination.
  • the method comprises introducing the recombination cassette into the recombination deficient host cell, and inducing the recombmantly deficient host cell to transiently support homologous recombination, thereby allowing the nucleic acid to integrate into the particular nucleotide sequence.
  • unselected nucleotide sequence rearrangements and deletions which are characteristic of host cells that support homologous recombination, are not evident with restriction endonuclease digestion map analysis with a restriction enzyme such as Hindlll, EcoRl, Xhol, or Avr ⁇ l.
  • unselected nucleotide sequence rearrangements and deletions are not evident with restriction endonuclease digestion map analysis with two or more restriction enzymes.
  • the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA .
  • inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of a RecA-hke protein in the host cell.
  • inducing the transient expression of the RecA-hke protein can be performed with a conditional replication shuttle vector.
  • the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
  • TSSV temperature sensitive shuttle vector
  • inducing the transient expression of the RecA-like protein comprises transforming the host cell with the TSSV at a permissive temperature, and growing the host cell at a non-permissive temperature.
  • the TSSV encodes a RecA-like protein that is expressed in the host cell and supports the homologous recombination between a nucleic acid contained in a recombination cassette and the particular nucleotide sequence contained in the host cell.
  • the TSSV encoding the RecA-like protein is diluted out when the host cell is grown at the non-permissive temperature.
  • the permissive temperature is 30°C and the non-permissive temperature is 43 °C.
  • the particular nucleotide sequence which has been selected to undergo homologous recombination is contained in an independent origin based cloning vector (IOBCV) that is comprised by the host cell, and neither the independent origin based cloning vector alone, nor the independent origin based cloning vector in combination with the host cell, can independently support homologous recombination.
  • IBCV independent origin based cloning vector
  • both the independent origin based cloning vector and the host cell are RecA "
  • inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of the RecA-like protein to support homologous recombination in the host cell.
  • the independent origin based cloning vector is a Bacterial or Bacteriophage-Derived Artificial Chromosome (BBPAC) and the host cell is a host bacterium.
  • BBPAC Bacteriophage-Derived Artificial Chromosome
  • inducing the transient expression of the RecA-like protein is performed with a conditional replication shuttle vector that encodes the RecA-like protein.
  • the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
  • TSSV temperature sensitive shuttle vector
  • the permissive temperature is 30°C and the non-permissive temperature is 43 °C.
  • the RecA-like protein is controlled by an inducible promoter and the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in the host cell.
  • the RecA-like protein is controlled by a constitutive promoter with the transient expression induced by the TSSV.
  • the TSSV also comprises a recombination cassette and a first gene which bestows resistance to a host cell that contains the TSSV against a first toxic agent.
  • the first gene can be counter-selected against.
  • the recombination cassette, the RecA-like protein gene, and the first gene are linked together on the TSSV such that when the nucleic acid integrates (i.e. resolved) into the particular nucleotide sequence, the RecA-like protein gene and the first gene remain linked together, and neither the RecA-like protein gene nor the first gene remain linked to the integrated nucleic acid.
  • the independent origin based cloning vector is a BBPAC and the host cell is a bacterium.
  • the BBPAC further contains a second gene that bestows resistance to the host cells against a second toxic agent.
  • Introducing the recombination cassette into the host cells is performed by transforming the host cell with the TSSV.
  • Inducing the transient expression of the RecA-like protein to support homologous recombination comprises: (i) incubating the host cells at a permissive temperature in the presence of the first toxic agent and the second toxic agent, wherein transformed host cells containing the TSSV and the BBPAC are selected for and wherein the RecA-like protein is expressed.
  • a first homologous recombination event occurs between the recombination cassette and the particular nucleotide sequence forming a co-integrate between the TSSV and the BBPAC, wherein the TSSV is either free or part of a co-integrate; (ii) incubating the transformed host cells at a non-permissive temperature in the presence of the first toxic agent and the second toxic agent, wherein host cells containing a TSSV co-integrate are selected for, and wherein free TSSV cannot replicate; (iii) selecting a host cell containing a co- integrate between the TSSV and the BBPAC by Southern analysis; (iv) incubating the host cells containing a co-integrate between the TSSV and the BBPAC at a non-permissive temperature in the presence of the second toxic agent, wherein a second homologous recombination event occurs between the recombination cassette and the particular nucleotide sequence, therein integrating the nucleic acid into the particular nucle
  • Another embodiment further comprises selecting a host cell containing the resolved BBPAC by colony hybridization with a labeled probe that binds to a DNA homologue of the nucleic acid, an mRNA homologue of the nucleic acid, and/or a protein encoded by the nucleic acid.
  • the permissive temperature is 30°C
  • the non-permissive temperature is 43 C C.
  • the incubating of host cells containing the resolved BBPAC in the presence of the second toxic agent and counter-selecting agent is performed at 37°C.
  • Preferred embodiments further comprise the generating of the recombination cassette by placing a first genomic fragment 5' of the specific nucleic acid that is to selectively integrate into the particular nucleotide sequence, and placing a second genomic fragment 3' of the specific nucleic acid.
  • the first genomic fragment corresponds to a region of the particular nucleotide sequence that is 5 ' to the region of the particular nucleotide sequence that corresponds to the second genomic fragment.
  • both the first genomic fragment and the second genomic fragment contain portions of the particular nucleotide sequence.
  • both the first genomic fragment and the second genomic fragment contain 250 or more basepairs of the particular nucleotide sequence.
  • the first and second genomic fragments are about the same size.
  • both the first genomic fragment and the second genomic fragment contain 500 or more basepairs of the particular nucleotide sequence. In still another embodiment, both the first genomic fragment and the second genomic fragment contain 1000 or more basepairs of the particular nucleotide sequence.
  • the recombination cassette is generated in a building vector and the recombination cassette is subsequently transferred to the TSSV.
  • the first gene confers tetracycline resistance and the counter- selecting agent is fusaric acid.
  • the RecA-like protein is recA.
  • the TSSV is pSVl. RecA having the ATCC no. 97968.
  • the RecA-like protein is controlled by an inducible promoter, and the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in the host cell.
  • the independent origin based cloning vector is a BBPAC and the recombination deficient host cell is an E. coli bacterium.
  • the RecA-like protein is recA.
  • the present invention also provides a conditional replication shuttle vector that encodes a RecA-hke protein.
  • the RecA- ke protein is controlled by an inducible promoter.
  • the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV).
  • the RecA-hke protein of the TSSV can be controlled by either a constitutive promoter or by an inducible promoter.
  • the TSSV contains a gene that can be counter-selected against.
  • the TSSV contains a gene that confers tetracycline resistance.
  • the TSSV contains a RecA-hke protein that is recA.
  • the TSSV contains both a gene that confers tetracycline resistance and a RecA- like protein that is recA.
  • the TSSV is pSVl.RecA having the ATCC no 97968.
  • the present invention also provides an independent origin based cloning vector that contains a particular nucleotide sequence that has undergone homologous recombination with a conditional replication shuttle vector in a RecA- host cell, wherein the conditional replication shuttle vector encodes a RecA-hke protein.
  • the particular nucleotide sequence is part of the gene that encodes the murme zinc fmger gene, RU49 which is contained by the independent origin cloning vector.
  • the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector m a RecA- host cell, wherein the temperature sensitive shuttle vector encodes a RecA-hke protein.
  • the independent origin based cloning vector is a BBPAC, and more preferably a BAC.
  • the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector that is pSVl .RecA having the ATCC no. 97968.
  • the present invention also provides methods of using the modified independent origin based cloning vectors of the present invention to make transgenic animals, perform gene targeting, or perform gene therapy.
  • the independent origin based cloning vectors or linearized nucleic acid inserts derived from the IOBCVs can be introduced into a eukaryotic cell or animal.
  • the eukaryotic cell is a fertilized zygote.
  • the eukaryotic cell is a mouse ES cell.
  • the gene targeting can be performed to modify a particular gene, or to totally disrupt the gene to form a knockout animal.
  • the independent origin based cloning vector contains a nucleic acid that has undergone homologous recombination with a conditional replication shuttle vector in a RecA " whole cell, in which the conditional replication shuttle vector includes a RecA like protein.
  • the independent origin based cloning vector is a BBPAC.
  • the BBPAC has undergone homologous recombination with a TSSV.
  • the BBPAC has undergone homologous recombination with the TSSV that is pSVl .RecA having the ATCC no. 97968.
  • One particular embodiment is a method of using the BBPAC to introduce the nucleic acid into an animal to make a transgenic animal comprising pronuclear injecting of the BBPAC (or a linearized nucleic acid insert derived from the BBPAC) into a fertilized zygote.
  • the animal is a mammal.
  • the mammal is a mouse.
  • the independent origin based cloning vector is a BBPAC and the fertilized zygote is a C57BL/6 mouse zygote.
  • two picoliters (pi) of less than one ⁇ g/ml BBPAC DNA is injected.
  • 2pl of 0.6 ⁇ g/ml of DNA is injected.
  • the present invention also includes a method of using the BBPAC of the invention to perform gene targeting in a vertebrate cells comprising introducing the BBPAC into the vertebrate cell wherein the nucleic acid that has undergone homologous recombination with the conditional shuttle vector, undergoes homologous recombination with the endogenous chromosomal DNA of the vertebrate cell.
  • the vertebrate cell is a mammalian cell.
  • the mammalian cell is a human cell.
  • the vertebrate cell is a fertilized zygote and the nucleic acid contains a disrupted gene.
  • the conditional shuttle vector is a TSSV.
  • the TSSV is pSVl.RecA having the ATCC no. 97968.
  • the present invention also contains kits for performing homologous recombination on selected nucleotide sequences contained on an independent origin based cloning vector, such as a BBPAC.
  • the kit comprises a conditional replication shuttle vector and a building vector.
  • the kit further contains a restriction map for the shuttle vector and/or a restriction map for one or more of the building vectors.
  • the kit further includes a protocol for using the contents of the kit to perform homologous recombination.
  • kits contains a TSSV, such as pSVl .RecA and a building vector.
  • the building vector is pBV.IRES.LacZ.PA.
  • the building vector is pBV.EGFPl .
  • the building vector is pBV.IRES.EGFPl .
  • the building vector is pBV.pGK.Neo.PA.
  • kits In a preferred embodiment two or more building vectors are included in the kit. In a more preferred embodiment all four of the above-listed building vectors are included in the kit. Restriction maps for one or more of the building vectors or the TSSV may also be included in the kits. In addition, the kits may also include a protocol for using the contents of the kit to perform homologous recombination. In one specific embodiment, a kit contains pS VI. RecA and one or more of the above-listed vectors also contains fusaric acid and/or chloro-tetracycline.
  • Figure 1 shows the strategy for targeted BAC modification.
  • Two cloning steps are involved in constructing the shuttle vector.
  • the recombination cassette (genomic fragments A and B; and IRES-LacZP-Poly A marker gene) is first constructed in the building vector and then subcloned into the temperature sensitive pSVLRec ⁇ shuttle vector.
  • Co-integrate formation Co-integrates can be formed through homologous recombination at either the homology A or the homology B site, with only the former case illustrated.
  • FIG 2 shows a schematic representation of targeted modifications of the BAC 169, which contains the mu ⁇ ne zinc finger gene, RU49.
  • BAC 169 containing RU49 was obtained from screening of the mouse 129 strain BAC genomic DNA library (Research Genetics).
  • Figure 2A depicts a restriction map of the BAC 169. The position of several exons are shown. The region of homology Al (lkb PCR fragment) and homology Bl ( ⁇ .6kb Xba-Hind fragment) are indicated.
  • Figure 2B depicts a map of the modified BAC 169 with IRES LacZ PolyA insertion (BAC 169. ILPA). An extra Pmel site is inserted with the marker gene (asterisk). The size of the two Pme-Not fragments and the Pmel fragment are indicated. Since the marker gene (4kb) is less than the deleted genomic region (7kb), the total size of the modified BAC (128kb) is smaller than the original BAC (13 lkb).
  • Figure 3 shows Southern blot analyses of BAC co-integrates and resolved BACs.
  • Figure 3 A shows a schematic representation of expected Southern blot fragments m BAC 169, in co- integrates through homology Bl, and in correctly resolved BACs.
  • an EcoRl digest is used and homology Bl is used as the probe;
  • a Hindlll digest is used and the homology Al is used as probe.
  • Figure 3B shows homology Bl co-integrates.
  • the EcoRl digest of BAC clones and controls are probed with homology B 1. 1-4 represent four clones. BAC 169 and pSVl with the recombination cassette were used as controls.
  • Figure 3C shows the analyses of the 5' ends of the resolved BACs.
  • Resolved BAC clones (1-8) were digested with Hindlll and probed with homology Al .
  • the controls are homology B 1 co-integrates (CI), BAC 169 and the shuttle vector with recombination cassettes.
  • Figure 3D shows the analyses of the 3' ends of the resolved BACs. The same procedure is used as desc ⁇ bed above except the resolved BAC clones were digested with EcoRl and probed with homology B 1.
  • Figure 4 shows pulsed field gel electrophoresis analyses of modified 169 with the ILPA insertion.
  • ILPA (LI and L2) and BAC169 were prepared by alkaline lysis, and then digested with Notl, Pmel and Xhol (in a standard buffer supplemented with 2.5 mM spermidme).
  • the digested DNA were separated by pulsed field gel electrophoresis (Bio-Rad's CHEF-DRII, 5 to 15s, 15 hours at 14°C) and blotted on to nitrocellulose filter (Stratagene). The same filter was probed separately with three probes.
  • LI and L2 are lacZl and LacZ2 which are independent clones which correspond to clones 1 and 2 respectively in Figures 3C and 3D.
  • Figure 4A shows the use of the BAC169 probe which revealed all the restriction fragments.
  • Figure 4B shows the use of the pgkpoly A probe which only hybridized to the ILPA insert fragment.
  • Figure 4C shows the use of the A2 probe which hyb ⁇ dized to a fragment outside the region of modification. The position of the markers are indicated.
  • Figure 5 shows the production of BAC transgenic mice.
  • Figure 5 A depicts purified linearized BAC LI 128 kb Not I insert for pronuclear injection.
  • the pulsed field gel is probed -with pgkpolyA probe.
  • the numbers represent different fractions.
  • the smear below the mtact fragment represent degradation and undigested DNA.
  • Figure 5B shows Southern blot analyses of the founder transgenic mice with the lacZ probe.
  • the tail DNA were digested with Bam HI and Southern blot analysis was performed.
  • the negative control consisted of httermates of Y3, Y7 and Y9 mice.
  • the positive control was a conventional transgenic mouse with the lacZ transgene.
  • Figures 5C and 5D show the results of using PCR to determine the presence of BAC ends in the transgenic mice.
  • the DNA at each end corresponding to the vector sequence is amplified and probed with a third ohgonucleotide in the middle of the fragment. The approp ⁇ ate size fragment is indicated.
  • the negative controls are httermates.
  • the positive control was BAC 169 DNA.
  • Figure 5E shows the germline transmission of the lacZ transgene m the Y7 mouse line. Tail DNA from two litters having eight mice each were prepared and digested with BamHl. Southern blot analysis was performed with the lacZ probe.
  • Figure 6 shows the expression of the lacZ transgene in the brain of the Y7 BAC transgenic line.
  • P6 mice brain from Y7 transgenic mice ( Figure 6A) and a wild type control litter mate ( Figure 6B) were whole mount stained to reveal lacZ expression m the Y7 cerebellum. Thick saggital sections (5mm) from Y7 transgenic mice were also stained for lacZ expression.
  • Figure 6C shows the low magnification
  • Figure 6D shows the high magnification of the rectangle area indicated in Figure 6C. Expression in the cerebellum, the detate gyrus and the lineage of the olfactory bulb are indicated (. e SVZ, RMS and the OB).
  • Ce cerebellum
  • SC superior colhcoli
  • IC mfenor colhcuh
  • DG dentate gyrus
  • VZ ventricular zone
  • SVZ subventncular zone
  • LV lateral ventricle
  • RMS rostral migratory tract
  • OB olfactory bulb
  • Co cortex
  • Figure 7 is a schematic diagram containing Figure 7A which depicts a hypothetical map of a gene of interest withm a selected BAC; Figure 7B which depicts the first targeted modification to introduce the positive selection marker gene; and Figure 7C which depicts the second modification to delete the promoter of the gene and to generate the short arm.
  • Figure 8 is the restriction map of pSVl.RecA. This temperature sensitive shuttle vector is based on the pMB096 vector ongmally constructed by M. O'Connor et al. [Science, 244:1307-1312 (1989)].
  • Figure 9 is the restriction map of pBV.IRES.LacZ.PA. This vector was modified from the pWHlO vector originally constructed by Kim et al. [MCB, 12:3636-3643 (1992)].
  • Figure 10 is the restriction map of pBV.EGFPl.
  • the plasmid is based on pBluesc ⁇ pt.KS(+).
  • EGFP1 was from Clonetech.
  • Figure 11 is the restriction map of pBV.IRES.EGFPl .
  • the plasmid is based on the pBluesc ⁇ pt.KS back bone.
  • EGFP1 was from Clonetech.
  • Figure 12 is the restriction map of pBV.PGK.Neo.PA.
  • the vector is based on a pBS.KS backbone.
  • the pGK.Neo.PA sequences was excised from a pKS.NT vector by digestion with Hindlll and BamHI and subcloned into the Hindlll/Bam fragment of the pBV.IRES.LacA.PA.
  • the present invention provides a simple method for directly modifying an independent origin based cloning vector (IOBCV) in recombination deficient host cells including generating deletions, substitutions, and/or point mutations in a specific gene contained in the independent origin based cloning vector. Such modifications may be performed with great specificity.
  • the modified independent origin based cloning vectors of the present invention can be used to introduce a modified heterologous gene into a host cell.
  • One specific use of such a modified vector is for the production of a germline transmitted independent origin based cloning vector transgenic animal.
  • Targeted independent origin based cloning vector modification can be used for functional studies in diverse biological systems.
  • the ability to efficiently modify a independent origin based cloning vector and generate an IOBCV-transgenic animal has important applications for functional analyses of genes in vivo.
  • modified independent origin based cloning vectors can be used to study regulation of genes or gene complexes in transgenic animals such as mice. Since modified independent origin based cloning vectors can be used to study gene function in vivo, a deletion, substitution and point mutation within a given gene can be made in a independent origin based cloning vector, and the independent origin based cloning vector containing the modified gene can be reintroduced in vivo in its endogenous expression pattern.
  • targeted independent origin based cloning vector modification can be used to create targeted expression of a selected gene, in the expression pattern of another gene, without prior knowledge of all of the regulatory elements of the selected gene.
  • An important application of this type is targeted expression of the ere recominase for tissue/cell type specific gene targeting [Kuhn et al, Science 269:1427 (1995); Tsien et al, Cell 87:1317 (1996)].
  • modified independent origin based cloning vectors can be used to generate large DNA constructs particularly for gene targeting in ES cells and in vivo.
  • the independent origin based cloning vector is a Bacterial Artificial Chromosome (BAC) modified in a host E.coli cell.
  • BAC Bacterial Artificial Chromosome
  • a targeted BAC modification system has several advantages over a conventional yeast based modification system. First, a modified BAC automatically returns to the recombination deficient state after modification, ensuring stable maintenance of the modified BAC m the host strain. Second, BAC DNA can be very easily purified in relatively large quantities and high quality, allowing for use in biological experimentation including pronuclear injection.
  • BAC libraries available from different species of animal, plants and microbes [Woo et al , Nucleic Acids Res , 22:4922 (1994); Wang et al , Genomics 24:527 (1994); Wooster et al , Nature 378:789 (1995)] Most BACs also include all the necessary regulatory elements (i.e. LCRs and enhancers) to obtain dose dependent and integration site independent transgene expression [Dillon et al Trends Genet 9.134 (1993), Wilson et al , Annu. Rev. Cell. Biol. 6.679 (1990); Bradley et al , Nature Genet 14.121 (1997)].
  • LCRs and enhancers i.e. LCRs and enhancers
  • Targeted BAC modification can be applied successively to dissect these elements.
  • a modified BAC may be used to generate a transgenic animal.
  • the BAC (or PAC) stably integrates into the animal cell genome.
  • the transgenic animal can be used for functional studies, or for generating a desired gene product, such as producing a human protein in the milk of a transgenic mammal [Drohan et al U.S. Patent No 5,589,604, Issued December 31, 1996].
  • modified BACs or PACs may be used for delivering a specific gene in gene therapy.
  • a modified BAC has been successfully inserted into a murme subject animal, and in vivo heterologous gene expression has been demonstrated.
  • the methodology of the present invention is very general. Whereas the targeted independent origin based cloning vector modification is demonstrated on BACs, the system is readily applicable to BBPACs in general including PACs, PI and other vectors propagated in the recombination deficient E coli.
  • the BAC modification exemplified herein is also apropo to Mammalian Artificial Chromosomes. For example, Harnngton et al [Nature
  • an "IOBCV” is an independent origin based cloning vector.
  • One example of such a cloning vector is a BBPAC defined below.
  • An IOBCV generally comprises a nucleic acid insert which either is or contains a gene of interest.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
  • a "Bacterial or Bacteriophage-Derived Artificial Chromosome” or “BBPAC” denotes a vector that is derived from a bacterium or bacteriophage such as a Bacterial Artificial Chromosome (BAC) which is an E. coli F element based cloning system, a Pi- Derived Artificial Chromosome (PAC) or a lambda-based cosmid.
  • BAC Bacterial Artificial Chromosome
  • PAC Pi- Derived Artificial Chromosome
  • the BBPAC encodes from 500 to 700 kilobases of genomic sequences.
  • the BBPAC encodes up to 500 kilobases of genomic sequences.
  • the BBPAC encodes between 120 to 180 kilobases of genomic sequences.
  • the BBPAC encodes 130 kilobases of genomic sequences.
  • a BBPAC used for gene targeting can be referred to as a "BBPAC targeting construct" and contains a nucleic acid insert
  • a “gene targeting construct” as used herein is used interchangeably with “targeting construct” and is a nucleic acid that when introduced into a cell undergoes homologous recombination with the endogenous chromosomal DNA of the cell.
  • the nucleic acid is introduced into the cell to induce a modification of a particular gene contained on the endogenous chromosomal DNA, including in particular cases, to disrupt that gene to create a knockout animal.
  • a recombmant deficient host cell is "RecA " " when the host cell is unable to express a RecA- ke protein, including recA itself, which can support homologous recombination
  • the gene encoding the RecA-hke protein has been deleted in a RecA host cell.
  • the RecA-host cell contains a mutation in the recA gene that impairs its function.
  • RecA-hke protein is defined herein to have the meaning generally accepted in the art except as used herein the recA protein itself is included as being a specific RecA-hke protein.
  • RecA- ke proteins are proteins involved in homologous recombination and are homologs to recA [Clark et al., Critical Reviews in Microbiology 20:125-142 (1994)].
  • the recA protein is the central enzyme in prokaryotic homologous recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination [McKee et al, Chromosoma 7:479-488 (1996)].
  • RecA-hke proteins have been found in eukaryotic organisms and yeast [Reiss et & ⁇ .,Proc.Natl.Acad.Sci. 93:3094-3098 (1996)] .
  • Two RecA-hke proteins in yeast are Rad51 and Dmcl [McKee et al (1996) supra].
  • Rad51 is a highly conserved RecA-hke protein m eukaryotes [Peakman et al, Proc.Natl.Acad Sci. 93:10222-10227 (1996)].
  • a "gene of interest” is a gene contained by a host cell genome or more preferably an independent origin based cloning vector that has been selected to undergo homologous recombination with a specific nucleic acid contained in a recombination cassette.
  • a gene of interest can be either specifically placed into the host cell or independent origin based cloning vector for this purpose, or already contained by the host cell or independent ongm based cloning vector .
  • a “marker” is an indicator, whose presence or absence can be used to distinguish the presence or absence of a particular nucleic acid and preferably the corresponding presence or absence of a larger DNA which contains and/or is linked to the specific nucleic acid.
  • the marker is a protein or a gene encoding the protein, and thus can be more specifically termed a “marker protein” or a “marker gene”.
  • marker (and thus marker protein or marker gene) is meant to be used extremely broadly and includes fluorescent proteins such as green fluorescent protein, enzymes such as luciferase, and further includes drug resistant proteins, whose presence or absence may not solely be regarded as a means to detect cells that contain the drug resistance protein; and/or he genes that encode such proteins.
  • drug resistance proteins and/or their corresponding genes can allow the preferential growth of cells that contain the drug resistant gene (or alternatively allow the counter-selection of cells that do not contain the drug resistant gene) and therefore bestow a type of selectable distinction which is meant to fall within the present definition of a marker.
  • a gene which encodes a marker protein is used herein interchangeably with the term “marker protein gene” and denotes a nucleic acid which encodes a marker protein.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • the present invention provides a recombination cassette that includes two homology fragments interrupted by an insertion, deletion or mutation sequence.
  • Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • nucleic acid molecule refers to the phosphate ester polymeric form of nbonucleosides (adenosme, guanosme, undine or cytidme; "RNA molecules”) or deoxy ⁇ bonucleosides (deoxyadenosine, deoxyguanosme, deoxythymidme, or deoxycytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, m either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the pnmary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter aha, in linear or circular DNA molecules (e g. , restnction fragments), plasmids, and chromosomes.
  • sequences may be descnbed herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscnbed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a “recombmant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • "Homologous recombination” refers to the insertion of a modified or foreign DNA sequence contained by a first vector into another DNA sequence contained second vector, or a chromosome of a cell.
  • the first vector targets a specific chromosomal site for homologous recombination.
  • the first vector will contain sufficiently long regions of homology to sequences of the second vector or chromosome to allow complementary binding and incorporation of DNA from the first vector into the DNA of the second vector, or the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 ' (ammo) terminus and a translation stop codon at the 3 ' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and franscnption termination sequence will usually be located 3 ' to the coding sequence.
  • Transc ⁇ ptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Withm the promoter sequence will be found a franscnption initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcnptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • a “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N- terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
  • the term "translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.
  • a particular nucleotide sequence comprising a gene of interest can be isolated from any source, particularly from a human cDNA or genomic library.
  • methods well known in the art, as described above can be used for obtaining such genes from any source (see, e.g. , Sambrook et al., 1989, supra).
  • any animal cell potentially can serve as the nucleic acid source for the molecular cloning of any selected gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences.
  • the present invention provides methods for selectively performing homologous recombination in a cell that normally cannot independently support homologous recombination.
  • a specific nucleic acid is inserted into a recombination cassette that selectively integrates into a particular nucleotide sequence when the recombination deficient cell is transiently induced to support homologous recombination.
  • the present invention allows the integration of a specific nucleic acid into a particular nucleotide sequence of a gene of interest.
  • the methods provided by the present invention minimize the nonspecific nucleotide sequence rearrangements and deletions, which are characteristic of other systems which involve host cells that normally support homologous recombination.
  • the specific nucleic acid can encode an entirely different protein than the gene of interest, and the gene of interest may be selected for the tissue specificity of its promoter, for example for use in generating a transgenic animal, or in a gene therapy protocol
  • the rat preproenkephahn gene may be used as the gene of interest since the preproenkephalm promoter has been shown to confer brain expression and synaptic regulation in transgenic mice [Donovan et al , Proc Natl Acad Sci 89.2345-2349 (1992)]
  • the mu ⁇ ne zmc finger gene, RU49 was used as the gene of interest.
  • the specific nucleic acid can be constructed so as to cause a deliberate and specific modification in the sequence of the gene of interest, for example for inducing a change in the ammo acid sequence of the gene product, such as is typically done in site-directed mutagenesis protocols
  • the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA .
  • alternative causes for recombination deficiency may be rectified by methods that are analogous to those taught by the present invention and/or readily apparent in view of such teachings
  • recombination deficiency may be due to a deficiency of an alternative recombination protein such as another Rec protein including recB, recC, recD, and recE [Clark et al , Critical Reviews in Microbiol 20.125-142 (1994)] which may be manipulated in a manner that is analogous to that taught herein for RecA-like proteins.
  • inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of a RecA-hke protein in the host cell
  • Such induction may be performed by expressing a RecA-hke protein contained by the recombination deficient host that is under the control of an inducible promoter
  • conditional replication shuttle vectors can also include pBR322 in a polyA temperature-sensitive bacterial strain
  • conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
  • TSSV temperature sensitive shuttle vector
  • Inducing the transient expression of the RecA-hke protein consists of transforming the host cell with the TSSV at a permissive temperature, and growing the host cell at a non- permissive temperature.
  • the TSSV encodes a RecA-hke protein that is expressed in the host cell and supports the homologous recombination between a specific nucleic acid contained m a recombination cassette and the particular nucleotide sequence contained in the host cell.
  • the TSSV encoding the RecA-hke protein is diluted out when the host cell is grown at the non-permissive temperature.
  • the particular nucleotide sequence which has been selected to undergo homologous recombination is contained by an independent origin based cloning vector (IOBCV) that is comprised by the host cell, and neither the independent ongm based cloning vector alone, nor the independent ongm based cloning vector m combination with the host cell, can independently support homologous recombination.
  • both the independent origin based cloning vector and the host cell are RecA " , and inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of the RecA-hke protein to support homologous recombination m the host cell.
  • the independent origin based cloning vector can be a BBPAC, such as the BAC exemplified below and the host cell can be a host bacterium, such as E. coli.
  • the independent origin based cloning vectors for use in the methods of the present invention can be obtained from a number of sources.
  • £.co//-based artificial chromosomes for human libraries have been descnbed [Shizuya et al, Proc. Natl. Acad. Sci. 89:8794-8797 (1992); Vietnamese et al, In Current Protocols in Human Genetics (ed. Dracopoh et al.) 5.15.1-5.15.24 John Wiley & Sons, New York (1996); Kim et al , Genomics 34:213- 218 (1996)].
  • BAC, PAC, and PI libraries are also available for a variety of species (e.g. Research Genetics, Inc., Genome Research, Inc., Texas A&M has a BAC center to make a BAC library for livestock and important crops). Also BACs can be used as a component of mammalian artificial chromosomes.
  • An independent origin based cloning vector that is a BAC can be isolated using a cDNA or genomic DNA probe to screen a BAC genomic DNA library, for example.
  • the use of a mouse genomic BAC library from Research Genetics is exemplified below.
  • a positive BAC can generally be obtained in a few days.
  • To insert a gene of interest into a selected locus in the BAC the region of insertion can be mapped for restriction enzyme sites. Whereas subcloning is necessary for detailed mapping, it is generally unnecessary since rough mapping is usually sufficient.
  • other independent origin based cloning vector genomic libraries can be screened and the isolated independent origin based cloning vectors manipulated in an analogous fashion.
  • conditional replication shuttle vectors of the present invention are constructed so as to contain a recombination cassette that can selectively integrate into the nucleotide sequence of the gene of interest encoded by the independent origin based cloning vector.
  • Such conditional replication shuttle vectors can be constructed by inserting a PCR amplified RecA- like gene into an appropriate conditional replication shuttle vector which either contains a specific drug resistant gene or can be subsequently modified to contain one.
  • the drug resistant gene can also be counter-selected against, such as with, tetracycline and fusaric acid.
  • the conditional shuttle vector can also contain a counter-selection gene such as a gene that confers sensitivity to galactose, for example.
  • the E.coli K12 recA gene (1.3kb) is inserted into the BamHl site of a pMB096 vector.
  • the vector already carried a gene that bestows tetracycline resistance, and in addition contains a pSClOl temperature sensitive origin of replication, which allows the plasmid to replicate at 30 degrees but not at 43 degrees.
  • the RecA-like protein of a conditional replication shuttle vector can be controlled by either an inducible promoter or a constitutive promoter.
  • the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in a host cell.
  • the constitutive promoter is the endogenous E. coli recA promoter.
  • the conditional replication shuttle vector should also contain at least one unique cloning site.
  • a building vector is used for the construction of the recombination cassette as described below, one unique site is reserved for transferring the recombination cassette containing the specific nucleic acid from the building vector to the conditional replication shuttle vector.
  • a polylinker can be inserted between two specific restriction sites to create additional restriction sites that allow cloning of the recombination cassette into the conditional replication shuttle vector.
  • the conditional replication shuttle vector created should minimally contain a recombination cassette comprising the specific nucleic acid, (e.g.
  • genomic fragments containing about 350 basepairs (e.g. 250 basepairs to 600 basepairs though less may be sufficient) or more of the gene of interest of the independent origin based cloning vector.
  • a building vector is used to construct the recombination cassette.
  • Two small genomic fragments each containing about 350 basepairs (e.g. 250 basepairs to 600 basepairs though less may be sufficient) or more of the gene of interest are cloned into the building vector (e.g., pBVl) in appropriate order and orientation to generate the flanking regions of the recombination cassette.
  • the recombination cassette is then transferred into the conditional replication shuttle vector (e.g., pSVl. RecA).
  • conditional replication shuttle vector is a TSSV and the TSSV is pSVl. RecA having the ATCC no. 97968.
  • conditional replication shuttle vector is transformed into a RecA " host cell containing the independent origin based cloning vector.
  • the independent origin based cloning vector can also contain a gene which bestows resistance to a host cell against a corresponding toxic agent/drug such as an antibiotic or in a specific embodiment, chloramphenicol.
  • the cells are grown under the conditions in which the conditional replication shuttle vector can replicate (e.g. , when the conditional replication shuttle vector is a TSSV which replicates at 30° but not at 43 °, the host cell is grown at
  • the transformants can be selected via the specific drug resistant gene (or first drug resistant gene) carried by conditional replication shuttle vector, and the second drug resistant gene carried by the independent origin based cloning vector.
  • conditional replication shuttle vector also carries the RecA-like protein gene, homologous recombination can occur between the conditional replication shuttle vector and the independent origin based cloning vector to form co-integrates through the sequence homology at either the 5' or the 3' flanking regions of the recombination cassette.
  • the co-integrates then can be selected by growing the cells on plates containing the first and second drugs at non-permissive conditions (e.g. for the TSSV above, at 43 °C) so that the non-integrated, free conditional replication shuttle vectors are lost.
  • the co-integrates can then be re-streaked onto plates containing the second drug, (i.e., the drug which the gene initially carried by the independent origin based cloning vector protects against) and grown under non-permissive conditions overnight.
  • the second drug i.e., the drug which the gene initially carried by the independent origin based cloning vector protects against
  • a fraction of the co- integrates undergo a second recombination event (defined as resolution), through sequence homology at either the 5' or the 3' flanking regions of the recombination cassette.
  • the resolved independent origin based cloning vector automatically loses both the first drug resistant gene (i.e., the specific drug resistant gene contained by the conditional replication shuttle vector) and the RecA-like protein gene due to the linkage arrangement of the RecA-like protein gene, the drug resistant gene and the specific nucleic acid on the conditional replication shuttle vector, described above.
  • the excised conditional replication shuttle vector cannot replicate under the non-permissive conditions and is therefore diluted out.
  • the resolved independent origin based cloning vectors can be further selected for by growing the host cells (e.g., at 37°C) on plates containing the second drug and an agent that counterselects against cells containing the gene resistant to the first drug (e.g., a gene conferring tetracycline resistance may be counter-selected against with fusaric acid).
  • the resolved independent origin based cloning vector will be either the original independent origin based cloning vector or the precisely modified independent origin based cloning vector.
  • One method to identify the correctly resolved BAC is to choose 5-10 colonies and prepare a miniprep DNA. The DNA can then be analyzed using Southern blots to detect the correct targeting events.
  • the desired clones can be identified by colony hybridization using a labeled probe for the specific nucleic acid contained by the recombination cassette.
  • probes are well known in the art, and include labeled nucleotides probes that hybridize to the nucleic acid sequence.
  • a marker nucleic acid can be included in the recombination cassette and constructed so as to remain with the specific nucleic acid upon integration into the independent origin based cloning vector.
  • the marker can be a marker gene or marker nucleic acid that encodes a marker protein that confers a specific drug resistance to the host cell, as exemplified above, against drugs such as antibiotics, e.g., ampicillin, chloramphenicol, and tetracycline, a protein that confers a particular physical characteristic to the cells, such as a green fluorescent protein or a modified green fluorescent protein as described in U.S. Patent 5,625,048, Issued 4/29/97 and WO 97/26333 Published 7/24/97 hereby incorporated by reference in their entireties, or an enzyme such as luciferase.
  • it can be another marker protein including e.g., ⁇ - galactosidase.
  • the methods of homologous recombination of the present invention are selective, and nonspecific nucleotide sequence rearrangements either do not occur, or are essentially undetectable by one or more conventional methods of analysis.
  • One such method includes pulsed field gel mapping of the modified independent origin based cloning vector and the unmodified independent origin based cloning vector to determine whether any unexpected deletions, or insertions or rearrangement were generated during the modification procedure.
  • the same filter can be probed separately with a probe for the whole independent origin based cloning vector, with a probe for the specific nucleic acid, and a probe for a region of the gene of interest that has not been modified.
  • a restriction enzyme digestion can reveal a finger print of the modified independent origin based cloning vectors indicating whether the fragments are preserved. Such a restriction enzyme digestion is exemplified below. Restriction enzyme digestions can be repeated with one or more additional restriction enzymes selected with respect to the restriction site map of the independent origin based cloning vector.
  • the modified independent origin based cloning vector and the unmodified independent origin based cloning vector can be assayed with both a probe specific for any region of the DNA contained by the recombination cassette predicted to be inserted into the independent origin based cloning vector (e.g., the promoter sequence, the specific nucleic acid, and a polyadenine addition signal sequence) and a probe specific for a region outside of the modification region (e.g., near the promoter region but outside of the modification region).
  • a probe specific for any region of the DNA contained by the recombination cassette predicted to be inserted into the independent origin based cloning vector e.g., the promoter sequence, the specific nucleic acid, and a polyadenine addition signal sequence
  • a probe specific for a region outside of the modification region e.g., near the promoter region but outside of the modification region.
  • a modified independent origin based cloning vector of the present invention can be purified by gel filtration, e.g. a column filled with SEPHAROSE CL-4B yielded intact linear BAC DNA.
  • the column can be pre-equilibrated in an appropriate buffer, as described in the Example below.
  • the purified DNA can be directly visualized with ultraviolet light after ethidium bromide staining, for example.
  • Columns such as the SEPHAROSE CL-4B column also can efficiently separate degraded DNA from the pure linear DNA.
  • the present invention also provides methods of using the modified independent origin based cloning vectors of the present invention.
  • modified independent origin based cloning vectors contain a nucleic acid that can be inserted into an animal to make a transgenic animal.
  • the modified independent origin based cloning vectors of the present invention can be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al, 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • Various therapeutic heterologous genes can be inserted into an independent origin based cloning vector of the invention such as but not limited to adenosine deaminase (ADA) to treat severe combined immunodeficiency (SCID); marker genes or lymphokine genes into tumor infiltrating (TIL) T cells [Kasis et al., Proc. Natl. Acad. Sci. U.S.A. 87:473 (1990); Culver et al., ibid. 88:3155 (1991)]; genes for clotting factors such as Factor VIII and Factor IX for treating hemophilia [Dwarki et al.
  • ADA adenosine deaminase
  • SCID severe combined immunodeficiency
  • marker genes or lymphokine genes into tumor infiltrating (TIL) T cells [Kasis et al., Proc. Natl. Acad. Sci. U.S.A. 87:473 (1990);
  • One particular method comprises the pronuclear injection of the modified independent origin based cloning vector into a fertilized animal zygote.
  • a method is exemplified below with the modified independent origin based cloning vector being a BAC which has been linearized, and the animal zygote being a mouse zygote. 2 pi of 0.6 ⁇ g/ml of BAC DNA was injected.
  • the presence of both ends of the modified independent origin based cloning vector can be assayed for in the transgenic animal to determine if the intact nucleic acid insert of the IOBCV has been integrated into the genome. Since both ends of the nucleic acid insert contain some vector sequence, PCR primers specific to the vector sequence can be generated and used to amplify the transgenic DNA. The amplified products can then be probed with a third labeled oligonucleotide probe within the amplified region.
  • transgenic animals that are formed give rise to germline transmission after appropriate breeding (B6/CBA mice were used in the Example).
  • the ratio of transgenic animals to wild type animals should follow Mendelian genetics.
  • the expression of the specific nucleic acid and/or the gene of interest inserted into the transgenic animal can be determined by a variety of methods well known in the art which depend on the nature of the insert. For example, enzymes can be appropriately assayed for activity, in the case of ⁇ -galactosidase, whole mount staining can be performed, in situ hybridization can be used to detect the corresponding mRNA, and specific antibodies can be used to identify the expression of a corresponding protein. In preferred embodiments such expression will be evident only in cells in which the endogenous gene of interest is expressed.
  • the present invention also provides the use of targeted BBPAC modification to obtain a high rate of gene targeting in vertebrates.
  • the BBPAC contains a nucleic acid insert comprising the gene targeting construct.
  • the circular BBPAC can be used, or preferably the linearized nucleic acid insert is used. In either case, the BBPAC or linearized nucleic acid insert can be purified by gel filtration as described herein.
  • the gene targeting is performed in ES cells using a BBPAC gene targeting construct that is greater than lOOkb.
  • the BBPAC gene targeting construct is similar to the conventional positive selection gene targeting construct ( Figure 7): it contains two regions of homology, a long arm ( > 80kb) and a short arm (10- 20kb), with the neo cassette (pgk-neo-polyA) introduced into the middle of the BBPAC.
  • the first modification is to introduce the neo gene to disrupt the gene of interest in the BBPAC.
  • a second modification is to create the short arm (10-20kb). The reason for the second modification is enable the use of an endogenous probe flanking the short arm (KO probe) to detect a polymorphism between the targeted allele and the wild type allele in screening ES cell clones (Fig 7; Gene Targeting, a practical approach, supra).
  • a preferred version of the BBPAC gene targeting methodology of the present invention also includes negative selection.
  • the conventional negative selection cassettes such as the use of the herpes thymidine kinase cassette or the diphtheria toxin gene cassette, may not always work with BBPAC constructs since BBPAC DNA tends to exist in transfected mammalian cells as episomal DNA for a long period of time [Baker et al. , NAR 25: 1950-1956].
  • the EGFP1 cassette can be used as a negative screening cassette.
  • the CMV promoter driven green fluorescent protein (EGFP-1) and the polyA signal can be introduced.
  • GFP is not toxic to the cells but serves as a fluorescent marker protein.
  • the EGFP-1 cassette When gene targeting occurs, the EGFP-1 cassette will be lost and the cell will not exhibit a green fluorescence under UV light.
  • the BBPAC integrates non-homologously, the EGFP-1 cassette also integrates, and the cells will therefore exhibit the green fluorescence under UV.
  • the definitive Southern blot analyses only those neo resistant cell lines which do not exhibit a green fluorescence under UV light are chosen.
  • the process of generating the targeted ES cells with a BBPAC targeting construct is essentially the same as with the conventional protocols (Gene Targeting, A Practical Approach, supra), except for the following steps.
  • the transfection of ES cells with the linearized intact BBPAC nucliec acid insert is performed as described by Baker [NAR, 25: 1950-1956 (1997)], using psoralen-inactivated adenovirus as carriers, for example.
  • the method enables transfection efficiency in mammalian cells with linear BBPAC DNA to be similar to the transfection efficiency of a conventional DNA construct.
  • the BBPAC targeting construct can potentially provide 10-100 fold higher targeting frequency than the conventional targeting construct, thereby making gene targeting in mouse ES cells easier and cheaper, since only a few dozen colonies need to be isolated and screened to obtain the targeted clones.
  • the present invention further provides a method of performing gene targeting in fertilized vertebrate zygotes by the injection of a BBPAC targeting construct, or preferably the linearized intact BBPAC nucleic acid insert containing the targeting construct to generate a transgenic knock-out animal (TKO).
  • a large targeting construct > lOOkb
  • TKO methodology has previously been attempted by Brinster et al.
  • the design of the gene targeting construct is similar to the ES cell targeting construct except that instead of the neo gene, an IRES-GFP cassette or an IRES- lacZ cassette is fused to an exon of the gene of interest to disrupt the gene ( Figure 7). As described above, two consecutive steps of BBPAC modifications are involved in generating the BBPAC containing the gene targeting construct.
  • the modified BBPAC TKO construct can be prepared in milligram quantities and linearized as described above.
  • the linearized DNA then is introduced into the fertilized zygote by a standard protocol, e.g., pronuclear injection (Hogan et al., (1986) supra).
  • the transgenic animal is then identified by standard Southern blots.
  • the gene targeting event can be further identified by digesting DNA of the transgenic animal with appropriate enzymes, such as enzyme X, ( Figure 7) and probed with the flanking KO probe ( Figure 7). Mice with the targeting event will have an additional band of the appropriate size.
  • Such gene targeting events can further be confirmed by expression of the GFP or LacZ marker gene in the expression pattern of the targeted endogenous gene, since the construct is designed to trap the endogenous promoter.
  • the TKO method has important ramifications in the field of vertebrate genetics. It enables gene targeting in many organisms that do not have ES cells, such as zebra fish, rats and other mammals. This will help to generate better animal models for human diseases (e.g. , rats and monkeys), or to create genetically targeted animals suitable for organ transplants (such as pigs or baboons) or for commercial reasons (e.g., leaner pork or beef).
  • This method also has additional advantages, even for gene targeting in mice. For example, this method will automatically provide germline transmission, since transgenic animals are rarely chimeric. It also enables targeted mice in strains other than the 129 strain to be obtained, and avoids the expensive and time-consuming out-breeding protocols.
  • BBPAC targeting constructs are provided. Since gene targeting in somatic cells is also dependent on the length of homology, using large DNA targeting construct also improves the targeting rate in somatic cells.
  • the experimental design in this case is similar to that with the ES cells described above. Somatic cell gene targeting is useful in gene therapy, for example, in a targeted insertion of a functional gene in a hereditary disease of the hematopoietic system. Such methods are also useful to generate targeted cell lines for experimental purposes.
  • conditional replication shuttle vectors that encode a RecA-like protein are also provided by the present invention.
  • the RecA-like protein can be controlled by either an inducible promoter or a constitutive promoter.
  • the conditional replication shuttle vector is preferably a temperature sensitive shuttle vector (TSSV).
  • TSSV temperature sensitive shuttle vector
  • the TSSV contains both a gene that confers tetracycline resistance and a RecA-like protein that is recA.
  • the TSSV is pSVl. RecA having the ATCC no. 97968.
  • independent origin based cloning vectors that contain a gene of interest that has been modified by the methods of the present invention are also included in the present invention. More particularly such independent origin based cloning vectors have undergone homologous recombination with a conditional replication shuttle vector in a RecA " host cell, wherein the conditional replication shuttle vector encodes a RecA-like protein. In a preferred embodiment the independent origin based cloning vector has undergone homologous recombination in a RecA " host cell with a temperature sensitive shuttle vector encoding a
  • the modified independent origin based cloning vector is a BAC that has undergone homologous recombination with the temperature sensitive shuttle vector pSVl.RecA having the ATCC no. 97968.
  • Bacterial based artificial chromosomes such as Bacterial artificial chromosomes (BACs) and P-lderived artificial chromosomes (PACs) are circular bacterial plasmids that may propogate as large as 300kb of exogenous genomic DNA (Shizuya et al, PNAS, 89, 8794- 97, 1992; Sicilnou et al, Nature Genet., 6, 84-90, 1994). For the majority of BAC and PAC libraries, the average size of the insert is 130-150 kb.
  • BACs Bacterial artificial chromosomes
  • PACs P-lderived artificial chromosomes
  • BAC and PAC libraries are much easier to construct due to higher cloning efficiency.
  • Second, BACs and PACs are propagated in recombination deficient E. coli host cells, so they have high stability and minimal chimerism. No rearrangements have been observed in BACs or PACs after 100 generations of growth.
  • Third, isolation of BAC and PAC DNA is very easy since they exist as supercoiled circular plasmids that are resistant to shearing. Conventional bacterial plasmid DNA isolation methods can be applied to obtain milligrams of intact BAC or PAC DNA.
  • Third, direct DNA sequencing can be applied to BAC or PAC DNA, which is not possible for YAC DNA.
  • BBPAC s are useful for physical mapping in genome studies, no simple method is available to modify BBPACs , as is available for the YACs.
  • a simple homologous recombination based BBPAC modification method is disclosed, termed targeted BBPAC modification (See Figure 7 for a schematic representation of the method). This method allows precise modification, such as marker insertion, deletion, point mutation, at any chosen site within a given BBPAC. This method involves several steps: isolation of
  • BBPACs using cDNA or genomic DNA probes, simple mapping and partial sequencing of the BBPACs, cloning of the shuttle vector, targeted modifications, pulsed field gel analyses of the modified BBPACs, and finally preparation of linearized BBPAC DNA for functional studies, such as pronuclear injection to produce BBPAC transgenic mice. Since the method is simple and reliable, it is reasonable to expect that the entire procedure, from the step of screening for a BBPAC with a cDNA or genomic DNA probe to the step of modified BBPACs ready for functional studies, can be completed within 6-8 weeks.
  • the IRES-LacZ marker gene has been introduced into an 13 lkb bacterial artificial chromosome (BAC) containing the murine zinc finger gene, RU49. No rearrangements or deletions are detected in the modified BACs. Furthermore, transgenic mice are generated by pronuclear injection of the modified BAC and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the cerebellum has been observed, which could not be obtained with conventional transgenic constructs. In summary, a novel and efficient method has been developed to modify BACs, PACs and PI for in vivo studies of gene expression and gene function.
  • BAC 13 lkb bacterial artificial chromosome
  • a BAC clone is isolated with either a unique cDNA or genomic DNA probe.
  • BAC libraries for various species (in the form of high density BAC colony DNA membrane) are available from Research Genetics, Inc. and Genome Research, Inc.
  • the mouse 129 genomic BAC library from Research Genetics has proved to be a good source for genomic DNAs.
  • the probe is first tested on a mouse genomic Southern blot to ensure that the probe does not contain any repetitive elements.
  • the library is screened according to manufacture's direction. The positive clones can be obtained from the company within a few days.
  • Solution I 50mM glucose, 25 mM Tris.HCl (pH 8.0); 10 mM EDTA (pH 8.0)
  • Solution II 0.2N NaOH, 1 % SDS (0.4 g NaOH, 45 ml ddH20, 5ml 10% SDS).
  • Solution III 5M KOAc (60ml), glacial acetic acid (11.5ml), H20 (28.5 ml).
  • the BAC midiprep DNA may be stored in 4 °C for months (Do not freeze the BAC DNA, since repetitive freezing and thawing will result in degradations).
  • (Ill) BAC maxiprep DNA preparation Two methods were used to prepare large quantities of RNA-free BAC maxiprep DNA.
  • the first method is the standard cesium chloride banding method (see Maniatis, supra).
  • This method was used routinely to obtain > 500ug BAC DNA from 1 liter bacteria culture.
  • the second method uses a commercially available column, the Nucleobond AX-500 (made by The Nest Group, Southborough, Mass.).
  • the maxiprep DNA are also stored in 4 °C for long-term storage.
  • the digested BACs are resolved on a pulsed field gel (Bio-Rad's CHEF-DRII).
  • the gel is 1 % agarose in 0.5 x TBE.
  • the gel is run in 0.5xTBE.
  • the separation condition is the following: 6v/cm, 5s to 15s linear ramping for 15hrs to 18hrs at 14 °C.
  • the New England Biolab's PFGE marker I or II as the high molecular weight marker and lkb DNA ladder (Life Technologies Inc.) as the low molecular weight marker are used.
  • the gel is then stained with ethidium bromide (1 to 5000, or 1 to 10,000 dilution of lOmg/ml stock) for 30 min prior to taking the photograph. Then the gel is blotted onto the nitrocellulose membrane and hybridized to cDNA and genomic DNA probes according to standard protocols (Maniatis, supra). To ensure the entire cDNA is included in the BAC, probes/or oligonucleotides from both the 5 'end and the 3' end of the gene are used to probe the blot separately. Those large BACs containing the entire gene are usually selected for BAC modification.
  • homologous sequence from the BAC has to be obtained.
  • Two homologous sequences of about 500bp each (namely A and B, Figure 7) is all that is needed to construct the shuttle vector for BAC modification.
  • the homologous sequences are chosen such that a given modification (i.e. insertion, deletion and point mutation) will be introduced between A and B in the BAC.
  • a and B can be obtained by direct sequencing of the BACs.
  • the sequencing oligonucleotides are designed based on the cDNA sequence.
  • step 2 If maxiprep DNA is used, go directly to step 2. If midiprep DNA is used, first add lOOul ddH20 and lOul lOmg/ml RNAse A to lOOul midiprep BAC DNA, and incubate at 37 °C for > lhr. (This step is critical, incomplete RNAse treatment will result in poor precipitation and sequencing).
  • Vectors used in targeted BAC modification A two vector system is designed to construct the shuttle vector for BAC modification
  • the first vector is a pBS.KS based building vector, which is used to construct the recombination cassette containing homologous sequence A and homologous sequence B and the modification to be introduced between them.
  • the recombination cassette was not constructed in the pS VI. RecA shuttle vector was for the following reasons: first, it is a low copy plasmid so that it is difficult to obtain high quantity DNA; second, it is a large plasmid (llkb), so it is relatively difficult to clone.
  • the building vector contains the marker gene to be introduced into the BAC, cloning sites flanking it (usually EcoRl for cloning the homology A and Xbal for homology B, and rare restriction sites such as Mlul, Pmel and Pac I for mapping of the modified BAC).
  • cloning sites flanking it usually EcoRl for cloning the homology A and Xbal for homology B, and rare restriction sites such as Mlul, Pmel and Pac I for mapping of the modified BAC.
  • the building vector One thing about designing the building vector is that there should not be any Not I sites within the recombination cassette, since Notl sites are used in the end to release the linear modified BAC for biological experiment (e.g., pronuclear injection).
  • Notl sites are used in the end to release the linear modified BAC for biological experiment (e.g., pronuclear injection).
  • the map and utility of various building vectors and the shuttle vector are described below.
  • pBV.IRES.LacZ.PA Building Vectors (pBV) All based on pBS.KS (Stratagene)
  • pBV.IRES.LacZ.PA Fig. 9
  • This vector is designed to introduce lacZ marker gene into a coding exon or the 3' UTR of a given gene, to study gene expression and gene regulation in vivo. IRES will enable the translation of the marker gene independent of the endogenous translation initiation codon.
  • pBV.EGFPl Fig. 10
  • This vector is designed to introduce the brighter version of the green fluorescent protein, EGFP1 (Clontech), into an exon of a given gene before the endogenous ATG or fused in frame with the endogenous gene.
  • the green fluorescent protein will mark gene expression in living cells and living organisms. Since the marker gene does not contain its own polyA addition sequence, the endogenous polyA sequence is used.
  • pBV.IRES.EGFPl (Fig. 11) This vector is used to introduce EGFP1 gene into the coding region or the 3' UTR of a given gene, with its translation independent of the endogenous translation frame.
  • pBV.pGK.Neo.PA This vector is designed to introduce a neo expression cassette into the BAC, containing the neo gene with the pgk promoter and the polyA addition signal.
  • Modified BAC can be introduced into tissue culture cell lines (i.e. ES cells) to obtain stable transfected cells by selecting for neomycin resistance.
  • This vector is particularly useful for gene targeting with modified BACs. Notice that although there are two identical pgkpA sequence at the 3' end of the neo gene, it will not interfere with the proper expression of the neo gene. The only consequence is that during BAC modification, one of the pgkPA sequence may be deleted due to homologous recombination.
  • the Sal I site is used to subclone the recombination cassette from the building vector.
  • the first step of targeted BAC modification involves the subcloning of two small genomic fragments (A and B) into an appropriate building vector, which includes two steps of conventional sub-cloning.
  • a and B small genomic fragments
  • PCR amplified fragment with appropriate restriction sites designed at the end of the PCR primer is the method of choice. Frequently, an additional restriction site is designed into one of the two PCR primers to assist in determining the orientation of the cloned PCR fragment. The relative imprecision of PCR amplification does not appear to affect the BAC modification efficiency.
  • Sal I is inactivated by heating to 65 °C for 15 minutes. 5.
  • the vector is then treated with alkaline phosphatase by adding 20ul lOx dephosporylaiton buffer, 4 ul (lunit/ul) calf intestinal alkaline phosphatase (Boehringer Mannheim) for 30 minutes at 37 °C.
  • the enzyme is then inactivated by adding 20 ul 50 mM EDTA (to a final concentration of 5mM), and heating at 75 °C for 15 minutes. 6.
  • the digested pSVl vector and pBV with recombination cassette are run on a 1 % low melting Seaplaque GTG agarose at 75 V for 8-10 hours.
  • the DNA should be run in a large well created by taping together several teeth of the comb.
  • Ligation reaction Each ligation reaction is done in 20ul total volume containing: >50ng pSVl. vector, 100-200ng insert, 2ul 10X ligation buffer (Boehringer-Mannheim), 2ul lOmM ATP, lul ligase (Boehringer-Mannheim) and ddH20. Ligation is carried out at 16 °C overnight.
  • Transformation of DH5a competent cells with pSVl vectors Half of the ligation reaction (10 ul) is used for transformation, by adding to 100 ul of cold, chemical- induced DH5a competent cells. Incubate 15 minutes on ice, then heat shock at 37 °C for 2 minutes, add 1ml LB to the tube, and shake at 30°C for 30 minutes. The cells are then centrifugated at 6000 x g for 4 minutes and the pellet is resuspended in 100 ul LB and spread onto Tet (lOug/ml) LB agar plates. Incubate the plates at 30 °C for > 15 hrs hours. 11.
  • Bacterial incubator set either at 30°C or at 43°C.
  • the following reagents and plates should be prepared prior to the targeted modification experiment. All the plates can be stored in 4 °C for up to one month. Detailed methods for preparation of various antibiotic resistant plates can be found in Maniatis.
  • Tetracycline stock solution 10 mg/ml in 50% ethanol, wrapped in aluminum foil and stored in -20°C for up to one month.
  • Tetracycline plates LB agar plates containing 10 ug/ml tetracycline. Store in 4 °C and wrapped in aluminum foil to avoid the light. 4. Chloramphenicol plates (Chi plates): LB plates contain 12.5 ug/ml
  • Tetracyline+ Chloramphenicol plates LB plates contain lOug/ml tetracycline and 12.5 ug/ml chloramphenicol.
  • Frozen stock of BAC containing DH10B cells were taken by a metal loop and inoculated into 3 ml of LB + chloramphenicol (12.5ug/ml). Grow the culture with rigorous shaking in 37 °C for overnight.
  • A) Two alternative metiiods can be used to identify the correctly resolved BACs. If bom A and B homology are about the same length, one can just pick 10-20 colonies, prepare miniprep DNA by alkaline lysis and do Soumern blot to analyze the targeting events. About half of me resolved BACs should contain the correctly targeted marker genes. B) If the two homology arms are not the same length (>500 bp difference), one should use the colony hybridization to select die correctly resolved BACs. Pick 50-100 individual colonies from FA+Chl plates, streak mem onto Chi plates and also onto the Tet+Chl plates, as a control for Fusaric acid selection.
  • Each plate can accommodate 50 test colonies and two positive control colonies, which are the co-integrate colonies from the Chi plate. Grow the colonies overnight at 37 °C. Abundant colonies should grow on die Chi plate, and none on the Tet-f- Chi plate, except the positive co-integrate controls.
  • the selection for tet sensitivity at step 4 is very stringent and has essentially no background. Therefore, all the colonies that grow on FA+Chl plates have been found to contain resolved colonies.
  • Colony hybridizations is performed, according to the standard protocols [Sambrook et al., (1989) supra], to select for the colonies that are resolved and resulted in targeted modification.
  • the colony hybridization probe should be part of the recombination cassette excluding the arms, such as lacZ, Neo, GFP or polyA sequences.
  • Midi-prep DNA are prepared for the positive clones by the alkaline lysis metiiod as described above. Restriction digests and Sou iern blots are performed to confirm targeting event on both homology side (A and B).
  • Pulse field gel analyses should be done to confirm die modification event and to determine if mere are any rearrangements in me modified BACs. Since there are two Not I site flanking the BAC insert (Research Genetics), digestion with Not I should reveal the size of die modified BAC. Generally Mlul, Pad and Pmel sites are included in the recombination cassette. Digestion with these enzymes will confirm the targeting events. Double digestion with these enzymes and with Not I will help to determine the integration site of die recombination cassette in the BAC. Xhol is usually used to fingerprint the modified BAC, since it has a wide distribution of fragment sizes. Comparing the Xho digestion pattern of die modified BAC with the original BAC will reveal any gross rearrangements in the modified BAC.
  • Od er enzymes such as BamHI and Avrll can also be used for this purpose.
  • Targeted BAC modification has been found not to introduce any unwanted rearrangements into the BACs.
  • Probes used to hybridized to die PFGE blots include: insert specific probes (s.a. lacZ, PolyA, GFP and Neo) and whole BAC probe (to reveal all d e digested bands from the BAC). Once the modified BACs are confirmed to have die specific targeted modification events and me lack of rearrangements, ti ese BACs are ready to be used for die biological experiments, such as producing transgenic mice or transfecting cells.
  • Purified DNA is stored at 4 °C. It is stable for weeks (e.g. , no degradation was detected after 3 weeks).
  • BACs are useful as tools for studying the regulation of gene expression in vivo.
  • a BAC can include the murine brain specific zinc finger gene, RU49 [Yang et al, Development 122:555 (1996)].
  • RU49 has been shown by in situ hybridization to be expressed in the granule cell population of the murine cerebellum, the dentate gyrus and the olfactory bulb in the brain.
  • proper expression of the lacZ marker gene could not be obtained in the cerebellum with a 10 kb RU 49 promoter-lacZ construct in transgenic mice, e.g., only one out often lines showed partial expression in the cerebellum.
  • the E.coli recA gene was introduced into the temperature sensitive shuttle vector.
  • the host strain becomes conditionally competent to perform homologous recombination allowing in vivo modification of the resident BAC.
  • Fig. 1 illustrates the steps involved in inserting a marker gene, e.g., IRES-lacZ-pGK polyA (ILPA), into the BAC.
  • a marker gene e.g., IRES-lacZ-pGK polyA (ILPA)
  • ILPA IRES-lacZ-pGK polyA
  • This shuttle vector is then transformed into E.coli containing the BAC.
  • the transformants can be selected by tetracycline resistance (carried by pSVl.RecA) and chloramphenicol resistance (carried by the BACs) at 30°C. Since the shuttle vector also carries the recA gene, homologous recombination can occur between the shuttle vector and the BAC, through either homology at A or B to form co-integrates. The co-integrates are selected by growth on tetracycline and chloramphenicol plates at 43 °C.
  • This temperature is non-permissive for shuttle vector replication, so that the non-integrated, free shuttle vectors are lost, resulting in the selection for bacteria carrying the integrated shuttle vectors, (either into the BACs or into the bacterial chromosomes). Correct BAC co-integrates can be identified by Southern blot analyses.
  • the co-integrates are then restreaked onto the chloramphenicol plates and grown at 43 °C overnight. A fraction of the co-integrates will undergo a second recombination event (resolution), through either homology at A or B.
  • the resolved BACs will automatically lose the tet and the recA genes, since the excised shuttle vector plasmids cannot replicate at the non-permissive temperature.
  • the resolved BACs can be selected by growing on chloramphenicol and fusaric acid plates at 37°C, as growth on fusanc acid plates selects for the loss of tetracycline resistance, i e , counterselectmg against BACs that are resistant to tetracycline.
  • the resolved BAC can be either the original BAC or the precisely modified BAC.
  • the desired clones can be identified by colony hybridization using a labeled probe for the inserted marker.
  • the recA gene is only temporally introduced into the bacterial host. Once the modification is finished, the bacteria will automatically lose the recA gene, returning to the recombination deficient state suitable for stable maintenance of the modified BACs.
  • This strategy termed targeted modification of BACs was tested by introducing the IRES- lacZ- polyA (ILPA) marker into the 131 kb murine BAC 169 containing the RU49 locus (Fig. 2A).
  • ILPA IRES- lacZ- polyA
  • the marker gene to the first coding exon of the RU49 gene was targeted with homology fragments being 1 kb and 1.6 kb respectively (Fig. 2B). Placing the IRES sequence before the lacZ gene ensures the translation of the marker gene even when lacZ gene is placed after the translation start site [Pelletier et al , Nature 334:320 (1988)].
  • the pSVl.RecA temperature sensitive shuttle vector containing the recombination cassette was transformed into the DH10 E coli strain containing the BAC 169 and selected by growth at either 30°C or 43 °C on plates containing chloramphenicol and tetracycline. In contrast to growth at 30 °C, which produced a thick lawn of transformed cells, growth at 43 °C resulted in growth of individual colonies. Twenty of these were picked and tested by Southern blots for co-integration of the shuttle plasmid into BAC 169. As shown m Fig.
  • Fig. 4 shows pulsed field gel mapping of the modified BAC LI and L2 and the original BAC 169.
  • the same filter was probed separately with the whole BAC 169 probe, with a probe from the inserted marker gene (pgkpolyA) and a probe from the 5 ' non-modified region of the RU49 gene (A2).
  • BAC169 probe (left panel) hybridizes with all the restriction fragments for each BAC.
  • Xhol digestion reveals a finger print of the modified BACs showing that essentially all fragments are preserved.
  • the fragment containing the ILPA insert is slightly smaller than the corresponding wild type fragment due to the replacement of the 7 kb RU49 fragment with the 4 kb marker gene (Fig. 2B).
  • Digestion with Notl which releases the entire BAC insert, also reveals a slightly smaller DNA insert in modified BACs for the same reason.
  • the marker gene was engineered to carry an additional Pmel sue, (Fig.2)
  • digestion of the BAC LI and L2 DNAs with this enzyme results in the generation of two fragments, in contrast to the single fragment seen in the original BAC169.
  • the sizes of these fragments allow the determination that these BACs contain approximately 75 kb 5' to the Pmel site, and 53 kb 3' to it (Fig. 2). No apparent rearrangements have occurred during the modification procedure.
  • both modified BACs and BAC 169 were probed with both a marker specific probe (pgkpolyA) and a probe near the promoter region and outside the modification region (A2). Consistently, both modified BACs contained a single band homologous to the marker gene probe which is not present in BAC 169. When the A2 probe was used, a single band of expected size appeared m all three BACs. Additional fingerprinting of all eight modified BACs with Hindlll, EcoRl and Avrll digests showed that no detectable rearrangements or deletion existed in these BACs. Thus, the temporary introduction of the recA gene into the BAC host strain does not introduce any rearrangements or deletions.
  • the BAC LI was further modified by replacing the IRES-lacZ sequence with pgk-neo sequence.
  • homologous fragments of about 500 bp each were used.
  • the modified BACs were also efficiently obtained and shown not to have any rearrangements or deletions. Therefore, targeted BAC modification is a simple method to precisely modify BACs without introducing any unwanted changes in the BACs.
  • transgenic mice carrying the modified BAC 169 with the IRES- LacZ insertion were generated.
  • To punfy the 128 kb BAC insert for pronuclear injection several established methods for purifying large YAC DNA were attempted, and resulted in considerable amount of DNA fragmentation.
  • a simple gel filtration column filled with SEPHAROSE CL-4B was tried, very pure fractions of mtact linear BAC DNA insert were obtained in an appropnate injection buffer, e.g., 100 mM NaCl, 10 mM T ⁇ s.HCl, pH 7.5 and 0.1 mM EDTA (Fig. 5A).
  • the purified fractions using the SEPHAROSE CL-4B column contained a large quantity of high concentration linear DNA (e.g., 0.5 mis of 3 ⁇ g/ml DNA or more).
  • the purified DNA could be directly visualized with ultraviolet light after ethidmm bromide staining.
  • the SEPHAROSE CL-4B column could also efficiently separate the degraded DNA (m this case in fractions 3-6) from the pure linear DNA (fractions 7-9) (F ⁇ g.5A).
  • Fraction 8 contained 3 ⁇ g/ml DNA and was used directly for pronuclear injection.
  • the Y7 transgenic mice also gave rise to germline transmission after breeding with B6/CBA mice. In two litters having a total of eight pups, three pups carried the LacZ transgene (Fig 5E). Further analysis demonstrated that the transgene was transmitted in a Mendelian distribution to more than fifty Y7 offspring.
  • lacZ gene in the cerebellum of the Y7 transgenic mice was determined by whole mount lacZ staining.
  • RU49 is normally expressed in the granule cells of the cerebellum, the dentate gyrus and the olfactory bulb (including the subventricular zone, the rostral migratory stream, and the olfactory bulb proper) [Yang et al, Development, 122:555-566 (1996)].
  • RU49 promoter lacZ transgenic mice with 10 kb promoter had been generated.
  • all of the transgenic lines showed strong positional effects: either they did not express in the brain at all, or they were ectopically expressed in the cortex, but not the cerebellum.
  • lOkb-lacZ transgenic line did show restricted expression in the cerebellum, however, the expression was restricted to the caudal half of the cerebellum.
  • the transgenic mice showed a lacZ expression pattern closely resembling the endogenous expression pattern (Fig. 6).
  • the marker gene is expressed throughout the cerebellum (Fig. 6A) and no expression is seen in five control httermates (Fig. 6B). Further analysis showed that the transgene is expressed at high level in the EGL and lower level in the IGL.
  • the lacZ marker gene is also expressed in the dentate gyrus and the rostral migratory stream and the olfactory bulb (Fig. 6C and 6D).
  • the pattern of the BAC transgene expression closely resembles the endogenous RU49 expression pattern in the brain. It is evident that the large genomic DNA in the BAC transgene can overcome the positional effects and confer the proper expression of RU49 in vivo, in contrast to our results using conventional transgenic constructs.
  • bacterial based artificial chromosomes are ideal for constructing large DNA for gene targeting.
  • BACs and PACs can be readily modified to introduce selection genes, marker genes, and deletions.
  • Making a BBPAC gene targeting construct will take about the same time as making a conventional targeting construct (1-3 months).
  • BBPAC targeting construct DNA can be easily isolated in milligram quantity and high quality. This is advantageous over the YAC system, since it is difficult to purify large quantities of high quality YAC DNA.

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Abstract

L'invention concerne un procédé simple destiné à modifier des gènes dans une cellule hôte présentant une déficience de recombinaison. De telles modifications consistent à produire une insertion, des délétions, des substitutions et/ou des mutations ponctuelles au niveau d'un quelconque site choisi dans le vecteur de clonage d'origine indépendante. Le gène modifié peut être contenu dans ce vecteur de clonage d'origine indépendante que l'on utilise pour introduire un gène hétérologue modifié dans une cellule. On peut employer un tel vecteur modifié dans la production d'un animal transgénique obtenu par lignée germinale, ou dans des protocoles de ciblage de gène dans des cellules eucaryotes.
PCT/US1998/012966 1997-06-23 1998-06-23 Procedes de preformage de recombinaison homologue d'apres une modification d'acides nucleiques dans des cellules presentant une deficience de recombinaison, et utilisation des produits d'acides nucleiques modifies de celles-ci WO1998059060A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU79848/98A AU730859B2 (en) 1997-06-23 1998-06-23 Methods of preforming homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof
JP50494899A JP2002515764A (ja) 1997-06-23 1998-06-23 組換え不能細胞において核酸の相同的組換えに基づく修飾を前成する方法及びその修飾した核酸産物の使用
CA002294619A CA2294619A1 (fr) 1997-06-23 1998-06-23 Procedes de preformage de recombinaison homologue d'apres une modification d'acides nucleiques dans des cellules presentant une deficience de recombinaison, et utilisation des produits d'acides nucleiques modifies de celles-ci
EP98930459A EP0998574A1 (fr) 1997-06-23 1998-06-23 Procedes de preformage de recombinaison homologue d'apres une modification d'acides nucleiques dans des cellules presentant une deficience de recombinaison, et utilisation des produits d'acides nucleiques modifies de celles-ci

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US08/880,966 US6143566A (en) 1997-06-23 1997-06-23 Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof
US10249098A 1998-06-22 1998-06-22
US09/102,490 1998-06-22
US08/880,966 1998-06-22

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JP (1) JP2002515764A (fr)
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WO2001004288A1 (fr) * 1999-07-09 2001-01-18 The European Molecular Biology Laboratory Procedes et compositions pour le clonage et le sous-clonage diriges utilisant la recombinaison homologue
WO2001005962A1 (fr) * 1999-07-20 2001-01-25 The Rockefeller University Recombinaison homologue conditionnelle de grands segments d'insertion de vecteur genomiques
WO2002002782A1 (fr) * 2000-06-29 2002-01-10 The Rockefeller University Activation de l'expression genique dans l'adn genomique eucaryote clone et procedes d'utilisation
US6485912B1 (en) 1997-06-23 2002-11-26 The Rockefeller University Methods of performing gene trapping in bacterial and bacteriophage-derived artificial chromosomes and use thereof
US6821759B1 (en) 1997-06-23 2004-11-23 The Rockefeller University Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof
WO2009114185A2 (fr) 2008-03-12 2009-09-17 The Rockefeller University Procédés et compositions pour profil translationnel et phénotypage moléculaire

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6485912B1 (en) 1997-06-23 2002-11-26 The Rockefeller University Methods of performing gene trapping in bacterial and bacteriophage-derived artificial chromosomes and use thereof
US6821759B1 (en) 1997-06-23 2004-11-23 The Rockefeller University Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof
WO2001004288A1 (fr) * 1999-07-09 2001-01-18 The European Molecular Biology Laboratory Procedes et compositions pour le clonage et le sous-clonage diriges utilisant la recombinaison homologue
US6355412B1 (en) 1999-07-09 2002-03-12 The European Molecular Biology Laboratory Methods and compositions for directed cloning and subcloning using homologous recombination
CN101492694B (zh) * 1999-07-09 2011-08-31 欧洲分子生物学实验室 应用同源重组定向克隆和亚克隆的方法和组合物
WO2001005962A1 (fr) * 1999-07-20 2001-01-25 The Rockefeller University Recombinaison homologue conditionnelle de grands segments d'insertion de vecteur genomiques
WO2002002782A1 (fr) * 2000-06-29 2002-01-10 The Rockefeller University Activation de l'expression genique dans l'adn genomique eucaryote clone et procedes d'utilisation
WO2009114185A2 (fr) 2008-03-12 2009-09-17 The Rockefeller University Procédés et compositions pour profil translationnel et phénotypage moléculaire
EP3369827A1 (fr) 2008-03-12 2018-09-05 The Rockefeller University Procédés et compositions pour profilage translationnel et phénotypage moléculaire

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CA2294619A1 (fr) 1998-12-30
AU730859B2 (en) 2001-03-15
EP0998574A1 (fr) 2000-05-10
JP2002515764A (ja) 2002-05-28
AU7984898A (en) 1999-01-04

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