WO2001005962A1 - Recombinaison homologue conditionnelle de grands segments d'insertion de vecteur genomiques - Google Patents

Recombinaison homologue conditionnelle de grands segments d'insertion de vecteur genomiques Download PDF

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WO2001005962A1
WO2001005962A1 PCT/US2000/019926 US0019926W WO0105962A1 WO 2001005962 A1 WO2001005962 A1 WO 2001005962A1 US 0019926 W US0019926 W US 0019926W WO 0105962 A1 WO0105962 A1 WO 0105962A1
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gene
shuttle vector
bac
protein
conditional replication
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Nathaniel Heintz
Xiangdong W. Yang
Shiaoching Gong
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The Rockefeller University
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • 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
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/204—Pseudochromosomes, minichrosomosomes of bacterial origin, e.g. BAC
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple 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.
  • high throughput methodology is provided for generating the modified the independent origin based cloning vectors.
  • 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.CellBiol 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. Set, 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.
  • BACs bacterial artificial chromosomes
  • PACs P-l derived artificial chromosomes
  • BACs are based on the E. coli fertility plasmid (F factor); and PACs are based on the bacteriophage PI.
  • F factor E. coli fertility plasmid
  • PACs 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 nonspecific and potentially deleterious recombination events are kept to a very minimum.
  • libraries of PACs and BACs are relatively free of the high proportion of chimeric or rearranged clones typical in YAC libraries, [Monaco et al, Trends Biotechnol 12:280-286 (1994); Boyseu et al, Genome Research, 7:330-338 (1997)].
  • isolating and sequencing DNA from PACs or BACs involves simpler procedures than for YACs, and PACs and BACs have a higher cloning efficiency than YACs [Shizuya et al, Proc. Natl. Acad.
  • Functional characterization of a gene of interest contained by a PAC or BAC 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 staining 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 in libraries relatively free of rearranged clones.
  • methodology for generating such cloning vectors there is also a need to apply such vectors to improve the results of the methods of gene transfer used in gene targeting, for creating animal models for diseases due to a dominant mutated allele, e.g., Huntington's disease, and for overexpressing in vivo proteins encoded by genes having an unknown function in order to determine the biological role of such genes.
  • 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). Using this specialized cell, mice can be generated with a targeted mutation [Joyner, A.
  • a major limitation for gene transfer procedures in vertebrate cells such as gene targeting is the low targeting frequency.
  • One critical factor affecting the targeting frequency is the total length of homology.
  • Deng and Capecchi [MCB, 12:3365-3371 (1992)] 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 ( ⁇ 10kb) targeting construct [Balasubramanian et al, J. of Bacteriology 178:273-279 (1996)]. Therefore, there is a need to construct large gene transfer constructs to allow efficient gene transfer in many biological systems.
  • the present invention provides a novel and an efficient high throughput method for 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 in a recombination deficient host cell, i.e., a cell that cannot independently support homologous recombination.
  • the method can employ 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 recombinantly 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 Avrll.
  • unselected nucleotide sequence rearrangements and deletions are not evident with restriction endonuclease digestion map analysis with two or more restriction enzymes.
  • a high throughput methodology is provided for generating modified independent origin based cloning vectors e.g. , BACs that comprise genomic DNA.
  • 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-like protein in the host cell.
  • inducing the transient expression of the RecA-like 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.
  • 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.
  • conditional replication shuttle vector contains an origin of DNA replication that requires the expression of a specific protein or proteins for replication that is (are) not normally present in host bacteria.
  • origin of DNA replication is the R6K ⁇ DNA replication origin [oriR (R6K ⁇ )] and the specific protein that is expressed by the specific host cell is the pi replication protein which is encoded by the pir gene.
  • 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.
  • conditional replication shuttle vector contains a TSSV that 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.
  • 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-like protein.
  • the RecA-like protein is controlled by an inducible promoter.
  • the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV).
  • the RecA- like protein of the TSSV can be controlled by either a constitutive promoter or by an inducible promoter.
  • the conditional replication shuttle vector contains an origin of DNA replication that requires the expression of a specific protein or proteins for replication that is (are) not normally present in host bacteria but is (are) in a specific host cell.
  • the conditional replication shuttle vector contains a gene that can be counter-selected against.
  • conditional replication shuttle vector contains a gene that confers tetracycline resistance.
  • conditional replication shuttle vector contains a RecA-like protein that is recA.
  • conditional replication shuttle vector contains both a gene that confers tetracycline resistance and a RecA-like protein that is recA.
  • conditional replication shuttle vector is a TSSV.
  • TSSV is pSVl. RecA having the ATCC no. 97968.
  • the present invention further provides conditional replication shuttle vectors that comprise an R6K ⁇ origin of replication and a nucleic acid encoding a recombination protein.
  • the recombination protein is recA.
  • the conditional replication shuttle vector is constructed so that it can modify a gene of interest in an IOBCV, preferably a BBPAC, and more preferably a BAC through homologous recombination. Such modifications include insertions, substitutions, and/or deletions.
  • the conditional replication shuttle vector further comprises a nucleic acid encoding one or more marker proteins or peptides that are to be inserted into the IOBCV so a particular gene product (encoded by the IOBCV) can be identified and/or monitored.
  • the nucleic acid encodes the marker protein IRES-EGFP.
  • the nucleic acid encodes the marker FLAG peptide.
  • the nucleic acid is taulacZ.
  • the nucleic acid is lacZ.
  • multiple maker proteins/peptides can be encoded in the conditional replication shuttle vectors of the present invention and subsequently inserted into/or adjacent to the protein encodeded by the gene of interest.
  • conditional replication shuttle vector further comprises a gene that can be counter-selected against.
  • the gene that can be counter-selected against is SacB.
  • the gene that can be counter-selected against confers tetracycline resistance.
  • conditional replication shuttle vector further comprises an A box region that either comprises or can be constructed to comprise a nucleic acid that can selectively integrate into a particular nucleotide sequence of a gene of interest contained by an IOBCV when the IOBCV and the conditional replication shuttle vector are placed in a host cell in which recombination events can occur.
  • the A box region is bracketed by two restriction enzyme sites.
  • the A box region and the restriction enzyme sites can be used to insert any selected nucleic acid into the conditional replication shuttle vector.
  • the two restriction enzyme sites are Ascl and Smal.
  • the selected nucleic acid is between 300 and 500 basepairs, though substantially larger nucleic acids can be used when desired.
  • conditional replication shuttle vector further comprises two fit sites.
  • the two frt sites are positioned on opposite sides of the A box. Since the frt sites are used in the resolution step following the co-integration of the selected nucleic acid with the IOBCV, when it is desired to place one or more markers into the IOBCV, these markers are also positioned in the conditional replication shuttle vector in between the two frt sites.
  • conditional replication shuttle vector further comprises two homologous nucleotide sequences, which are homologous to each other, but preferably are not homologous to the IOBCV that comprises the nucleotide sequence which forms the co-integrate with the selected nucleic acid of the conditional replication shuttle vector.
  • the homologous nucleotide sequence is longer than the corresponding selected nucleic acid.
  • the homologous nucleotide sequence is greater than 500 basepairs.
  • homologous nucleotide sequence is greater than 1000 basepairs.
  • homologous nucleotide sequence is greater than 5000 basepairs.
  • the two homologous nucleotide sequences are positioned on opposite sides of the A box. Again, since the two homologous nucleotide sequences are used in the resolution step following the co- integration of the selected nucleic acid with the IOBCV, when it is desired to place one or more markers into the IOBCV, these additional markers are also positioned on the conditional replication shuttle vector in between the two homologous nucleotide sequences.
  • the two homologous nucleotide sequences preferably encode one or marker proteins (and peptides).
  • the homologous nucleotide sequence encodes the enhanced green fluorescent protein (IRESEGFP).
  • the present invention provides methods of selectively performing homologous recombination with a particular nucleotide sequence of an independent origin based cloning vector (IOBCV) that is contained in a recombination deficient host cell.
  • Such methods comprise introducing a conditional replication shuttle vector into a recombination deficient host cell and therein enabling homologous recombination in the host cell via the transient expression of a recombination protein in the host cell.
  • the host cell comprises an IOBCV which contains the particular nucleotide sequence whereas the conditional replication shuttle vector encodes a recombination protein that is transiently expressed by the host cell.
  • conditional replication shuttle vector also contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed. Neither the IOBCV alone, nor the IOBCV in combination with the host cell can independently support homologous recombination.
  • the present invention further provides methods of selectively modifying a particular nucleotide sequence of an independent origin based cloning vector (IOBCV) that is contained in a recombination deficient host cell that are particularly conducive for high throughput procedures.
  • IBCV independent origin based cloning vector
  • One such embodiment comprises introducing a conditional replication shuttle vector into a recombination deficient host cell in which the host cell contains an IOBCV that comprises a gene of interest which contains a particular nucleotide sequence.
  • the conditional replication shuttle vector encodes a recombination protein that is expressed by the host cell and permits homologous recombination to occur in the host cell since neither the IOBCV alone, nor the IOBCV in combination with the host cell can independently support homologous recombination.
  • the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA " .
  • the recombination protein is the rec E and rec T protein pair.
  • the recombination protein is the Lambda beta protein. In yet another embodiment the recombination protein is the Arabidopsis thaliana DRT100 gene product. Preferably, the recombination protein is rec A.
  • the IOBCV is preferably a BBPAC and more preferably the BBPAC is a BAC.
  • the conditional replication shuttle vector contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed, thereby forming a co-integrate.
  • the nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the recombination protein are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the recombination protein remains with the conditional replication shuttle vector.
  • conditional replication shuttle vector cannot replicate dilutes out the conditional replication shuttle vector encoding the recombination protein, and thereby prevents further (undesirable) recombination events in the recombination deficient cells to occur.
  • the conditional replication shuttle vector further comprises a nucleic acid that encodes a marker protein or peptide.
  • the nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the marker protein or peptide are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the marker protein or peptide is inserted into or adjacent to the particular nucleotide sequence.
  • the conditional replication shuttle vector cannot replicate in the host cell because the conditional replication shuttle vector requires a particular protein for replication, and neither the host cell nor the IOBCV encode the particular protein.
  • the conditional replication shuttle vector cannot replicate in the host cell because the conditional replication shuttle vector comprises a R6K ⁇ origin of replication and neither the host cell nor the IOBCV encode pir.
  • conditional replication shuttle vector further comprises a first frt site that is positioned on one side of the nucleic acid that selectively integrates into the particular nucleotide sequence, and a second frt site that is positioned on the other side of the nucleic acid that selectively integrates into the particular nucleotide sequence.
  • the resolution of the co-integrate is performed by adding flip recombinase to the host cell.
  • Flip recombinase is preferably added to the host cell by introducing a plasmid that encodes flip recombinase to the host cell.
  • the plasmid contains a conditional origin of replication such as a temperature-sensitive origin of replication which allows the plasmid to be diluted out by growing the host cells at a temperature that disfavors the replication of the plasmid.
  • the conditional replication shuttle vector can further comprise a nucleic acid encoding one or more marker proteins and/or peptides that are positioned in between the two frt sites and are also adjacent to the nucleic acid that selectively integrates into the particular nucleotide sequence, such that after the resolution, the marker protein(s) and/or peptide(s) are contained by the IOBCV.
  • the resolution step can be performed by a second homologous recombination step.
  • the conditional replication shuttle vector further comprises two homologous nucleotide sequences that are homologous to each other but are not homologous to the IOBCV.
  • the two homologous nucleotide sequences are positioned on the conditional replication shuttle vector to be on opposite sides of the nucleic acid that selectively integrates into the particular nucleotide sequence so that the resolution of the co-integrate is performed by a recombination event between the two homologous nucleotide sequences.
  • the two homologous nucleotide sequences are used in the resolution step following the co-integration of the selected nucleic acid with the IOBCV, when it is desired to place one or more markers into the IOBCV, these additional markers are also positioned on the conditional replication shuttle vector in between the two homologous nucleotide sequences.
  • the two homologous nucleotide sequences preferably encode one or marker proteins.
  • the homologous nucleotide sequence encodes the enhanced green fluorescent protein (e.g., IRESEGFP).
  • a more preferred embodiment further comprises adding a counterselection agent after the resolution of the co-integrate to remove host cells that comprise the conditional replication shuttle vector.
  • the conditional replication shuttle vector is designed to further comprise a counterselection gene that is positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate the counterselection gene remains with the conditional replication shuttle vector.
  • the counterselection gene is SacB.
  • the counterselection agent is sucrose.
  • the present invention also provides the 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 of the present invention.
  • the conditional replication shuttle vector encodes a RecA-like protein.
  • the particular nucleotide sequence can be all or part of a given gene such as the gene that encodes the murine zinc finger gene, RU49 (also known as Zipro 1) as exemplified below.
  • the nucleotide sequence can be constructed to further contain specific translational or transcription elements such as an IRES, and/or marker proteins such as the green fluorescent protein.
  • the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector in a RecA- host cell, wherein the temperature sensitive shuttle vector encodes a RecA-like 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 including making animal models for diseases due to a dominant mutated allele, e.g. , Huntington's disease; perform gene targeting; perform gene therapy; or for overexpressing in vivo proteins encoded by genes having an unknown function in order to determine the biological role of such genes, as exemplified below.
  • the independent origin based cloning vectors or linearized nucleic acid inserts derived from the IOBCVs for example, can be introduced into a eukaryotic cell or animal.
  • the transgenic animal made has a particular phenotype as a result of introducing (e.g., by pronuclear injecting) a BBPAC into the transgenic animal (or a fertilized zygote) which corresponds to a symptom of a particular disease.
  • a BBPAC had been modified to contain a dominant allele known to be associated with and/or due to the particular disease.
  • a BBPAC is identified that contains the wildtype copy of a gene that has been associated with one or more symptoms of a particular disease when the nucleotide sequence of the gene has a particular modification.
  • the BBPAC containing the wildtype gene is modified through homologous recombination by a method of the present invention, e.g. with a conditional replication shuttle vector, so that it contains the nucleotide sequence that has been associated with one or more symptoms of the particular disease.
  • the modified BBPAC is then placed into a transgenic animal or a eukaryotic cell (e.g. , a fertilized zygote) which results in a transgenic animal that has a phenotype that can be correlated with one or more symptoms of the particular disease.
  • the transgenic animal can then be used as an animal model for the particular disease.
  • the eukaryotic cell is a fertilized zygote.
  • the eukaryotic cell is a mouse ES cell.
  • the gene targeting for example, can be performed to modify a particular gene, or to totally disrupt the gene to form a knockout animal.
  • IOBCVs made by the methods disclosed herein can be added in multiple copies to a fertilized mammalian zygote for example, in order to achieve overexpression of a particular protein.
  • an IOBCV made by the methods disclosed herein can be used to make an animal model for a particular disease in which the expression of a mutated allele (carried by the IOBCV) leads to the desired phenotype for the animal model.
  • 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 replication 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 replication shuttle vector is a TSSV.
  • the TSSV is pSVl .RecA having the ATCC no. 97968.
  • the IOBCVs (including BBPACs and BACs) that have been modified by the methods of the present invention are also part of the present invention.
  • the present invention further provides methods of producing non-human transgenic animals using these IOBCVs.
  • One such method comprises introducing the IOBCV into a eukaryotic cell and placing the eukaryotic cell into a recipient animal, whereby the eukaryotic cell develops into the non-human transgenic animal.
  • the eukaryotic cell is a fertilized animal zygote.
  • the eukaryotic cell is an embryonic stem cell.
  • the eukaryotic cell is an ES- like cell.
  • all of the non-human transgenic animals generated by such methodology are also part of the present invention.
  • 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.
  • an independent origin based cloning vector such as a BBPAC.
  • Any of the shuttle vectors of the present invention can be included in the kits.
  • 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.
  • a particular embodiment of the kit 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.
  • two or more building vectors are included in the kit.
  • 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.
  • kits may also include a protocol for using the contents of the kit to perform homologous recombination.
  • a kit contains pS VI. Rec A 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 pSVl J?ec4 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 murine 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 (1.6kb Xba-Hind fragment) are indicated. Abbreviations: Xhol (Xh), EcoRl (R), Hindlll (H), Xbal (X), Notl (Not) and Pmel (Pme).
  • Figure 2B depicts a map of the modified BAC 169 with IRES LacZ Poly A insertion (BAC169.
  • ILPA 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 (131kb).
  • 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 in BAC 169, in co-integrates through homology B 1 , 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 Bl. 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 Bl 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 described above except the resolved BAC clones were digested with EcoRl and probed with homology Bl .
  • Figure 4 shows pulsed field gel electrophoresis analyses of modified 169 with the ILPA insertion.
  • ILPA (LI and L2) and BAC 169 were prepared by alkaline lysis, and then digested with Notl, Pmel and Xhol (in a standard buffer supplemented with 2.5 mM spermidine).
  • the digested D ⁇ A 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 BAC 169 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 hybridized 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 pgkpoly A probe. The numbers represent different fractions. The smear below the intact 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 littermates 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.
  • FIG. 5E shows the germline transmission of the lacZ transgene in 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 in 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 (i.e. SVZ, RMS and the OB).
  • Ce cerebellum
  • SC superior collicoli
  • IC inferior colliculi
  • DG dentate gyrus
  • VZ ventricular zone
  • SVZ subventricular 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 within 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 pS VI. Rec A. This temperature sensitive shuttle vector is based on the pMBO96 vector originally 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 pWHIO 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 pBluescript.KS(+).
  • EGFP1 was from Clonetech.
  • Figure 11 is the restriction map of pBV.IRES.EGFPl.
  • the plasmid is based on the pBluescript.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.
  • Figures 13a-13f show the targeted disruption of Ru49 gene.
  • Figure 13a is a schematic drawing of the targeting vector which contains a 3.7 kb Hz ' «dlil-Hzwdlil and a 6 kb BamH -Xbal fragments as the arms. The neo gene replaces the first two coding regions. Restriction sites are abbreviated as follows: B; BamHl, ⁇ ; Hindlll, R; EcoRl, X; Xbal.
  • Figure 13b shows the Southern blot analysis of a litter obtained from a heterozygous cross; tail DNA digested with BamHl and probed with 5' fragment shown in Figure 13a.
  • Figures 13c,and 13d show the P20 midsagittal cerebellar paraffin sections that were stained with cresyl violet at P20.
  • Figures 13e, and 13 f are mitotic cells shown using immunohistochemistry using antibody to phosphorylated histone 3 on midsagittal sections of P9 cerebella. Representative positive cells indicated by arrows in -/- ( Figure 13 e) and +/+ ( Figure 13/).
  • Figure 14 contains a schematic drawing of the relevant regions of the BAC 169, BAC169.fEGFP and BAC169.ILPA. and fine restriction mapping of these BACs.
  • a part of BAC 169 containing the Ru49 exon 3-5 are shown ( Figure 14 a).
  • the corresponding region containing the modification made in BAC169.tEGFP ( Figure 14 b) and BAC169.ILPA ( Figure 14 c) are also shown.
  • the open box represents the untranslated region of an exon.
  • the closed box represents the coding region of the exons.
  • the location of restriction sites for Hindlll ( ⁇ ) and EcoRl (R) are indicated.
  • BACs were digested with EcoRl (lanes 1- 3) or H dlll (lanes 4-6) and probed with the 1.6kb Xba-Hind probe ( Figure 14 ) or with the 131 kb BAC 169 probe ( Figure 14e).
  • Figures 15a-15g show the generation of BAC169tRGFP transgenic mice.
  • Figures 15a displays the Southern blot analysis of four BAC169t ⁇ GFP transgenic lines (Fl mice) and two wildtype mice using an IRES. EGFPl probe.
  • Figures 15b displays the Northern blot analysis of Ru49 expression in the cerebella of P10 Dl and E6 transgenic mice and wildtype littermates.
  • Figure 15 c the same Northern blot filter in Figure 15b was probed with an IRES.EGFPl probe.
  • Figures 15d displays the Western blot of cerebella from P7 transgenic and wildtype mice of the E6 that were probed with M2 Flag antibody.
  • Figure 15e shows that direct inspection under epifluorescence reveals EGFPl expression in the BAC169tEGFP transgenic cerebellum. EGFPl is not observed in the adjacent pons and brainstem (BS).
  • Figure 15f shows that on thick cerebellar sections (100 ⁇ M), EGFPl is expressed in the EGL, the IGL and the molecular layer (ML). But, it is not expressed in the Pukinje cell layer (PC).
  • Figure 15g shows the Histochemical analysis of P7 LacZ fresh frozen sagittal sections. Expression is highest in the EGL but can be detected in the IGL as well.
  • Figures 16a-16f show the morphological alterations in the BAC169tEGFP transgenic cerebella.
  • Figure 16a shows the P12 transgenic and wildtype cerebella (CE). The width of the wildtype cerebellum is indicated by the bar.
  • Figure 16b is a sagittal section of a P20 transgenic cerebellum.
  • Figure 16c is a sagittal section of a wildtype P20 cerebellum.
  • Figure 16d is a camera-lucida drawing of the posterior surface of a transgenic cerebellum, indicating the foliation pattern including three intralobular fissures: CrIF, CrIIF and PMDF.
  • Figure 17a- 17f contrast cell proliferation vs cell death in the BAC169tEGFP transgenic mice.
  • the arrows indicate cells positively labeled using the TUNEL method, reflecting an approximate twofold increase in cell death in the IGL.
  • Figure 17e shows a bar chart depicting 3 H-thymidine incorporation assays with P8 cerebellar granule cells. The absolute incorporation values from one of the four independent experiments is shown.
  • Figures 18a-18d show the genetic influence of the Ru49 gene dosage on the formation of four intralobular fissures in the cerebellum at P20-P22. The number of animals used for each measurement (n) is indicated. The statistical significance was measured using the ⁇ 2 analysis. The asterisk indicates a P-value of less than 0.001.
  • Figures 19a-19h show the skin phenotype ⁇ Ru49 transgenic mice versus wildtype mice.
  • Figure 19a is a photograph of two mice showing the appearance of a BAC169.tEGFP transgenic mouse with alopecia at P20 (right) and a wildtype littermate (left).
  • Figure 19b shows tails of E6 transgenic (top) and wildtype(bottom) mice at P9 that are viewed under epifluoresencent microscope.
  • Figure 19c depicts the LacZ histochemical staining of the whole mount skin of BAC 169. ILPA (Y7) transgenic mouse (left) and wildtype littermate (right).
  • Transgenic (Figure 19d) and wildtype mouse ( Figure 19e) skin sections were stained with cresyl violet.
  • Figure 20 depicts a conditional replication shuttle vector that comprises a gene encoding Rec A, a gene encoding the enzyme levansucrase (sacB), the R6K ⁇ origin of replication, the two restriction sites Ascl and Smal, an "A” Box, nucleic acids encoding an enhanced green fluorescent protein (EGFP) and the epitope tag (FLAG), tau lacZ and two FRT sites.
  • sacB levansucrase
  • FLAG epitope tag
  • Figure 21 depicts a Southern blot demonstrating the efficacy of performing transient homologous recombination to modify a BAC using the vector of Figure 20. Eight of the ten colonies picked for analysis contained the desired product.
  • Figure 22 depicts a schematic drawing of a procedure for using a conditional replication shuttle vector to modify a BAC, culminating in using the modified BAC to make transgenic mice.
  • This procedure includes the expression of flp recombinase within the cells containing the cointegrate to excise the shuttle vector sequences [Hoang et al, Gene 212:77-86 (1998)].
  • the flp recombinase works via the "frt" sites surrounding the shuttle vector.
  • Figure 23 depicts a conditional replication shuttle vector that comprises a gene encoding Rec A, a gene encoding the enzyme levansucrase (sacB), the R6K ⁇ origin of replication, an "A" Box, tau lacZ, a nucleic acid encoding the epitope tag (FLAG) and two copies of a nucleic acid encoding an enhanced green fluorescent protein (EGFP).
  • sacB levansucrase
  • tau lacZ a nucleic acid encoding the epitope tag
  • FLAG epitope tag
  • EGFP enhanced green fluorescent protein
  • Figure 24 depicts the conintegrate of the shuttle vector of Figure 23 and the BAC.
  • Figure 25 depicts a Southern blot monitoring the final resolution step using the conditional replication shuttle vector of Figure 23. 11 of 17 colonies tested yielded the desired product (arrow,), 5 others have correctly resolved cointegrates that resolved back through the "A box” to give the original unmodified BAC. No DNA was recovered in the final sample (lane 9).
  • the present invention provides a simple method for directly modifying an independent origin based cloning vector (IOBCV) in recombination deficient host cells including generating insertions (such as adding markers), 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 placement into ES cells, ES-like cells [Cibelli et al, Nature Biotechnology 16:642- 646 (1998); Pain et al, Cells Tissues Organs, 165:212-219 (1999)] or 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 in 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 there are many more 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, including as an animal model for a disease, 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.
  • modified BACs have been successfully inserted into murine subject animals, and in vivo heterologous gene expression has been demonstrated.
  • Example 2 a modified BAC construct was constructed so that the C-terminus of the gene product of the gene of interest was fused with two in- frame epitope tags and the gene of interest was further appended to an IRES/EGFP marker gene.
  • similar modifications can be performed so that the N-terminus of the gene product of the gene of interest comprises one or more markers.
  • the present methodology allows any portion of the BAC DNA to be altered/modified and therefore also allows such modifications/alterations/deletions at any site of the gene product of the gene of interest.
  • the methods of the present invention are fully amenable to modifications, alterations, fusions and the like to selected genes of interest and/or portions thereof (e.g., the coding regions) and furthermore can be successfully employed for generating animals with desired genotypes and/or phenotypes.
  • genetic analysis in mice has most commonly employed two general strategies: phenotypic screens for spontaneous or induced mutations; and genotypic analysis using homologous recombination or gene trapping to produce deletion or insertion mutants.
  • genetic analysis in invertebrates has recently emphasized over- or misexpression studies to understand gene function, the use of increased gene dosage analysis in mice has been hampered by variability in the expression patterns and levels of most conventional transgenes.
  • bacterial artificial chromosome (BAC) mediated gene dosage analysis in transgenic mice can be employed to reveal novel genetic functions that are not evident from conventional loss-of- function mutations.
  • Ru49 zinc finger transcription factor 1
  • Zipro 1 zinc finger transcription factor 1
  • Ru49 is also found to be expressed in the skin, and increased Ru49 gene dosage results in a hair loss phenotype that is associated with increased epithelial cell proliferation and abnormal hair follicle development.
  • the methods disclosed herein can also be used to correctly express dominant negative or gain-of-function mutations via BAC mediated transgenesis that offer additional avenues for genetic analysis in a selected animal (e.g. , a mouse or a monkey).
  • the present invention further provides a simple and rapid method for modifying and then resolving IOBCVs (e.g, BACs) in E. coli which is useful for large scale modification of BACs.
  • One such method employs a shuttle vector that comprises a conditional origin of replication (e.g., the R6K ⁇ DNA origin of replication), a nucleic acid encoding a recombination protein, (e.g., recA,) to induce the host cell to support homologous recombination, and a positive counter-selection marker, (e.g., the SacB gene which allows the selection for resolved BAC clones by sucrose).
  • a conditional origin of replication e.g., the R6K ⁇ DNA origin of replication
  • a nucleic acid encoding a recombination protein e.g., recA,
  • a positive counter-selection marker e.g., the SacB gene which allows the selection for resolved BAC clones by sucrose.
  • the procedure is performed by a high throughput method which allows the modification of the IOBCV is liquid and allows the efficient resolution of the vector.
  • 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, Harrington et al. [Nature Genetics, 15:345-355 (1997)] have used BAC derived DNA as a component of their Human Artificial Chromosome. Therefore, the use of such human artificial chromosomes can include the BAC modification taught by the present invention.
  • an "IOBCV” is an independent origin based cloning vector.
  • 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 “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 recombinant deficient host cell is "RecA " " when the host cell is unable to express a RecA-like protein, including recA itself, which can support homologous recombination.
  • the gene encoding the RecA-like 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-like 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- like protein.
  • RecA-like proteins are proteins involved in homologous recombination and are homologs to recA [Clark et al., Critical Reviews in Microbiology 20:125-142 (1994)].
  • the rec A 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-like proteins have been found in eukaryotic organisms and yeast [Reiss et al.,Proc.Natl.Acad.Sci. 93:3094- 3098 (1996)] .
  • Two RecA-like proteins in yeast are Rad51 and Dmcl [McKee et al. (1996) supra].
  • Rad51 is a highly conserved RecA-like protein in eukaryotes [Peakman et al, Proc.Natl.Acad.Sci. 93:10222-10227 (1996)].
  • a "recombination protein” as used herein is a protein involved in homologous recombination that can be used either alone or in conjunction with other proteins to allow homologous recombination to proceed in a cell that is otherwise recombination deficient.
  • recombination proteins include RecA-like proteins, the rec E and rec T proteins which are encoded by the Rec E gene [Clark et al, J.Bacteriol 175:7673-7682 (1993); Hall et al, J. Bacteriol 175:277-287 (1993); Kusano et al.
  • 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 origin 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 the 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 encode 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 ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in 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 primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5 ' to 3 ' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the rnRNA).
  • a "recombinant 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 in 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 ' (amino) 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 rnRNA, 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 transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional 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.
  • a transcription 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 transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into rnRNA, 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.
  • fusion protein and “fusion peptide” are used interchangeably and encompass “chimeric proteins and/or chimeric peptides” and fusion "intein proteins/peptides".
  • a fusion protein of the present invention comprises at least a portion of the protein or peptide encoded by a gene of interest of the present invention joined via a peptide bond to at least a portion of another protein or peptide in a chimeric/ fusion protein.
  • fusion proteins can comprise a marker protein or peptide, or a protein or peptide that aids in the isolation and/or purification of the protein or peptide encoded by a gene of interest of the present invention.
  • heterologous nucleotide sequence is a nucleotide sequence that can be covalently combined with a gene of interest of the present invention (e.g., by homologous recombination) to modify the gene of interest.
  • Such nucleotide sequences can encode chimeric and/or fusion proteins.
  • the heterologous nucleotide sequence can also encode peptides and/or proteins which contain regulatory and/or structural properties.
  • a heterologous nucleotide sequence can encode a protein or peptide that can function as a means of detecting a protein or peptide encoded by a gene of interest (contained by a BAC, for example).
  • a heterologous nucleotide sequence can function as a means of detecting a nucleotide sequence of the present invention.
  • a heterologous nucleotide sequence can also comprise non-coding sequences including restriction sites, transcriptional regulatory elements, promoters and the like.
  • 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, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al. , 1989”)] .
  • 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 preproenkephalin gene may be used as the gene of interest since the preproenkephalin 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 murine zinc 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 amino 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-like protein in the host cell.
  • Such induction may be performed by expressing a RecA-like 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.
  • 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 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-like protein that is expressed in the host cell and supports the homologous recombination between a specific 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 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 origin based cloning vector alone, nor the independent origin based cloning vector in 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-like protein to support homologous recombination in 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.
  • E.coli-based artificial chromosomes for human libraries have been described [Shizuya et ⁇ l, Proc. Natl. Acad. Sci. 89:8794-8797 (1992); Vietnamese et ⁇ l., In Current Protocols in Human Genetics (ed. Dracopoli 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 replication 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 pMBO96 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 rec A 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 preferably 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 transfeoed into the conditional replication shuttle vector (e.g., pSVlJ?e ⁇ 4).
  • the recombination cassette, the RecA-like protein gene, and the drug resistant gene are linked together on the conditional replication shuttle vector such that when the specific nucleic acid integrates into the particular nucleotide sequence, the RecA-like protein gene and the drug resistant gene remain linked together, and neither the RecA-like protein gene nor the drug resistant gene remain linked to the integrated specific nucleic acid.
  • the 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.
  • 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 30°C) and 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. Since the 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. This results in the selection for host cells carrying the integrated conditional replication shuttle vectors, (which co- integrate either into the independent origin based cloning vector or into the host chromosome). Correct independent origin based cloning vector co-integrates can be identified by PCR or more preferably with Southern blot analyses.
  • 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.
  • 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 (the disclosures of which are 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 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 1 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.
  • 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, hpofection (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 BAC can be introduced into an embryo which is then transplanted into a recipient animal.
  • 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 1).
  • 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.
  • Example 1 in which the gene of interest was the murine zinc finger RU49, and the specific nucleic acid inserted therein was the lacZ marker gene, analyses of the expression of the lacZ marker gene in the entire cerebellum of postnatal day 6 transgenic mice closely resembled the corresponding endogenous RU49 expression pattern.
  • Example 2 RU49 was epitope-tagged with FLAG and His, and coexpressed with enhanced green fluorescent protein using an IRES.
  • 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.
  • Two targeted BBPAC modifications are used to make this construct. 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 EGFPl 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-homo logously, 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 adeno virus 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 (>100kb) can provide a very high targeting rate (predicted by mathematical modeling described above) and gene targeting can be directly performed with a fertilized vertebrate zygote via pronuclear injection of the modified BBPAC targeting construct.
  • TKO methodology has previously been attempted by Brinster et al. [PNAS, 86:7087-91 (1989)] with a small DNA construct (2.6-8.9kb) but those workers only obtained a relatively low targeting rate (0.2%).
  • the large homology DNA in the BBPAC (>100kb) of the present invention increases the targeting rate to a favorable range of 2% to 10%.
  • 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).
  • 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).
  • 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.
  • the present invention further provides alternative methods of utilizing BBPAC transgenic analysis for the characterization of specific genes and their encoded protein products.
  • ESTs are selected from mammalian UniGene sets.
  • the EST clones are used for the identification of BBPAC clones (preferably BAC clones) containing the genes of interest.
  • the gene expression and protein localization in selected animals can then be analyzed and these data can then be tabulated.
  • Specific BAC/EGFP or ⁇ -lactamase constructs for example, can also be used to prepare cell specific probes for gene expression analysis using chips or arrays. The isolation of these cell types can be achieved using fluorescence activated cell sorting (FACS).
  • cDNA probes can also be prepared from these cell types and they can be hybridized to cDNA arrays or chips. The application of these methods can be also used to characterize cell specific changes in gene expression in selected biological paradigms or transgenic models of human disease.
  • the present invention also includes the use of marker insertion into
  • BBP ACs clones and transgenic analysis to precisely map the patterns of expression of tissue (or system, such as the CNS) specific genes including through determining the localization oftheir protein products.
  • the method can include the utilization of the human and mouse UniGene projects as informatics engines for the identification of genes that are predominantly expressed in a particular tissue or system.
  • the UniGene databases compile "sets” or “clusters” of EST sequences to identify those representing a single gene, and collate information about these genes into an easily accessible online database.
  • the human project has incorporated EST sequences from 150 cDNA libraries prepared either from brain tissue or CNS derived cell lines, and organized these data in the "Genome Anatomy Summary" according to sites of expression of each cluster of cDNAs.
  • the “Digital Differential Display (DDD)” project is a "computational method for comparing sequence-based gene representation profiles among individual cDNA libraries” and it results in the classification of genes present in these libraries into useful categories.
  • DDD Digital Differential Display
  • the present invention further provides methods preparation of transgenic mice with the modified BBP ACs of the present invention.
  • 4 founders for each BAC are generated so that the expression patterns can be analyzed at el 3, P0, P9, and adult stages. Data for the P9 and adult stages can be obtained directly from founders since they can be identified prior to these ages. Data for el 3 and PO mice will require one round of breeding to generate Fl animals.
  • the founder(s) chosen for breeding are preferably males to maximize the yield of Fl progeny.
  • pregnant females can be sacrificed, embryos typed and analyzed.
  • P0 animals from additional litters can also be sacrificed, typed and analyzed.
  • At least two transgenic Fl progeny from each strain can be allowed to age to detect any apparent phenotypes due to increased gene dosage due to integration of multiple copies of the BAC (see Example 2, below).
  • the BBP ACs obtained can be used in many ways including to make further modifications (e.g., ere insertion, or generation of dominant negative mice, etc.) or to isolate specific cell types and/or to characterize cell types in slice preparations
  • the present invention further provides methods of analysis of marker gene expression patterns and localization of epitope tagged protein products.
  • visualization of the marker gene expression pattern is to perfuse in 4% paraformaldehyde, dissect the brain, postfix in 4% para, cryoprotect in 10%) PVP, 4% sucrose, freeze in OCT, section at 40 microns, and float sections on PBS for direct visualization or processing for immuno fluorescence. Direct visualization is effective for GFP, as is immunofluorescence with ⁇ GFP antibodies. If the expression level is very low, it is sometimes advantageous to amplify the signal using immunoflourescence.
  • the sections can be processed for immunofluorescence with the myc epitope tag f, double immunofluorescence or direct visualization combined with immunofluorescence for the myc tag.
  • vectors using ⁇ -lactamase as the marker gene can be constructed to assess whether this is advantageous for detection.
  • the sections chosen for analysis should be optimized to obtain the most information in the least number of images.
  • One procedure entails collecting lateral, midsaggital and medial sections from each developmental stage and recording digital images for each of these time points. Images can be saved at both low and high magnifications to record the generalized expression pattern, the morphology of cells expressing the marker, and the localization of the epitope tagged protein in individual cells.
  • One half of a brain can be used to run a Western blot to determine the size of the epitope tagged protein.
  • the epitope tag is placed on the carboxyl-terminal amino acid.
  • the epitope tag can also be placed in other positions of the protein being expressed.
  • certain classes of proteins will not be properly localized if the epitope tag is fused to the C-terminus, e.g., the terminal three amino acids of many receptors are critical for interaction with scaffolding proteins carrying PSD domains [Korneau et al, Science 269:1737-1740 (1995)]. In this case insertion of the epitope tag just N-terminal to these crucial C-terminal amino acids can be performed.
  • 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), though for large scale procedures, the use of the R6K ⁇ DNA replication origin along with the pir replication protein may be used instead [see above].
  • conditional replication shuttle vector contains both a gene that confers tetracycline resistance and a RecA-like protein that is recA.
  • conditional replication shuttle vector is a TSSV such as the 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
  • the independent origin based cloning vector has undergone homologous recombination in a RecA " host cell with a temperature sensitive shuttle vector encoding a RecA-like protein.
  • 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.
  • the present invention further provides methods of generating animal models for diseases associated with and/or due to a dominant allele.
  • One such model is for Huntington's disease which has recently been generated with a YAC construct [Hodgson et al, Neuron 23:181-192 (1999)].
  • the BBP ACs modified by the methods of the present invention are superior to the YACs since the BBP ACs of the present invention can be generated with a higher cloning efficiency, have a higher stability, and have minimal chimerism.
  • Such an animal model can be generated by placing BBPAC into an animal zygote, wherein that BBPAC contains a nucleic acid that has undergone homologous recombination, in a RecA " host cell, with a conditional replication shuttle vector that encodes a RecA-like protein. Since the BBPAC can contain the entire gene encoding a particular protein, (which depending on the particular animal model desires can comprise a particular mutation), the gene can be expressed in the cells of the animal model that it is normally expressed in the disease.
  • a mutation is identified in a gene that has been linked to a particular disease.
  • a BBPAC library is screened for the wildtype gene (e.g., with a nucleic acid probe, or by computer searching). The precise alteration/modification of the gene is performed by a homologous recombination procedure disclosed herein using a conditional replication shuttle vector of the present invention.
  • the resulting modified BBPAC is isolated and then placed into an animal thereby forming the animal model (e.g., injecting into the nucleus of a zygote).
  • the BBPAC further comprises a marker so as to readily identify animals that contain the BBPAC.
  • conditional replication shuttle vector is a TSSV.
  • the TSSV is pSVl.RecA having the ATCC no. 97968.
  • the nucleic acid is introduced into the animal by pronuclear injecting the BBPAC into a fertilized zygote and thereby forming the animal model.
  • Any dominant allele can be used to generate the corresponding animal model for the disease.
  • Such dominant alleles include but in no way is limited to: huntingtin (htf) involved in Huntington's Disease [Hodgson et al, Neuron 23:181-192 (1999)]; PKD2 involved in polycystic kidney disease [Makowitz et al, Am. J. Physiol. 277:F17-F25 (1999)]; CACNA1A involved in Familial hemiplegic migraine [Carrera et al, Neurology53 :26-33 (1999)]; the RP1 gene involved in retinitis pigmentosa [Guillonneau et al., Hum. Mol Genet 8:1541-1546 (1999)]; and presenilin-1
  • the BBPAC comprises a mutant huntingtin (htt).
  • Any non-human animal can be used for the animal model, including standard laboratory rodents such as mice, rats, rabbits, and guinea pigs; farm animals such as sheep, goats, pigs, and cows; and higher primates such as monkeys, and the great apes such chimpanzees and gorillas.
  • the non-human animals are also part of the present invention.
  • the present invention further provides a method of preparing modified IOBCVs, e.g., BBP ACs that can be used in high throughput procedures.
  • modified IOBCVs e.g., BBP ACs
  • Such high throughput procedures are invaluable for gene mapping, for example. Indeed, such high throughput procedures can be used to readily generate high resolution atlases of gene expression in specific organs and tissues which involve thousands or even tens of thousands of genes.
  • one aspect of the present invention allows the labeling of specific gene products including placing epitope tags and or visually detectable labels (e.g., green fluorescent protein, LacZ, and tau-LacZ) on the gene products.
  • labeling allows the expression of the gene to be monitored.
  • the present invention permits phenotypic analysis, see Example 2.
  • the high throughput procedures disclosed herein also provide a means to rapidly and reliably increase the gene copy number of a large number of individual genes by rapidly generating the appropriate BACs. Such methodology is particularly helpful when studying a specific pathway, disease and/or organ or tissue.
  • this methodology permits archiving such modified BAC constructs for local reinjection and regeneration of a particular transgenic animal rather than warehousing expensive animal strains (e.g., transgenic mice) for long periods in a central facility.
  • the vector contains a protein-dependent origin of replication, e.g., pLD55, which comprises the R6K ⁇ origin of replication.
  • the R6K ⁇ origin is completely dependent on the pir gene, which is not carried in the BAC strains [Metcalf et al, Plasmid 35:1- 13 (1996), the contents of which are hereby incorporated by reference in its entirety; Shizuya et al, PNAS, 89:8794-8797, (1992)]. Therefore, cloning into the shuttle vector can be carried out in apir+ bacterial strain in which the shuttle vector can be propagated effectively.
  • the R6K ⁇ origin of replication allows growth at a high copy number in strains that express the pir protein, i.e., contain the pir gene. This is advantageous both because it is very simple to obtain large amounts of DNA for cloning into this vector and because the plasmid cannot persist on its own in the BAC strain.
  • a nucleic acid encoding a recombination protein, e.g., the recA gene, can be inserted into the pLD55 forming the pLD55.recA, for restoration of homologous recombination in the BAC strain as disclosed above.
  • conditional replication shuttle vectors that have a R6K ⁇ origin of replication are not replicated during or following the homologous recombination step between the conditional replication shuttle vector and the BAC. This is because the R6K ⁇ origin of replication has an absolute requirement for pir to replicate and the homologous recombination step takes place in the BAC strains, which do not express pir. This means that there is no further replication of the conditional replication shuttle vectors that comprise a R6K ⁇ origin of replication in BAC strains. In fact, it was surprising that these shuttle vectors could successfully support transient homologous recombination without replicating in the cells in which the homologous recombination was occurring.
  • conditional replication shuttle vectors having a temperature-sensitive origin of replication, since a small but significant percentage of temperature-sensitive vectors that can replicate at 30 °C and supposedly cannot replicate at 43 °C, still replicate at 43 °C.
  • the lack of independent shuttle vector replication is important since it significantly increases the percentage of cells that will comprise the vector-BAC cointergrate, after the cells are grown under conditions that require the presence of both the BAC and the shuttle vector. Indeed, this high efficiency makes it practical to modify BACs as disclosed herein, in liquid media rather than on plates. Furthermore, it is the use of a liquid media that makes it possible to modify numerous different BACs at one time.
  • the Ori R6K ⁇ conditional replication shuttle vector takes advantage of an analogous selection system as disclosed herein for the temperature sensitive conditional replication shuttle vector, but is far more preferable for liquid media high throughput procedures.
  • the vector also comprises the tet R and amp R selectable markers.
  • tet R tet R and amp R selectable markers.
  • a more robust tet R gene is used since the allele present in the original pLD55 vector was not optimal for the fusaric acid negative selection that is used in the resolution step of the BAC modification procedure, yielding pLD55.recA.tet.
  • This vector thus carries both the recA gene and selectable markers used in the BAC modification protocol disclosed herein, and in a particular embodiment merely substitutes the original temperature-sensitive plasmid origin taught herein with the conditional R6K ⁇ origin [Metcalf et al, Plasmid 35:1-13 (1996)].
  • this shuttle vector When this shuttle vector is electroporated into the BAC strains, as stated above, it absolutely cannot replicate since the BAC strains do not express the pir protein. Thus, the only way the BAC strain can contain both the chloramphenicol resistance and the tetracycline resistance markers (other than do to potential background, see below) is if the shuttle vector integrates into the BAC episome forming the cointegrant that is sought (the BACs contain a chloramphenicol resistance gene).
  • the first is dependent on the efficiency of the negative selection for the conditional replication shuttle vector, i.e., whereas the R6K ⁇ origin of replication yields a background of 10 "8 , the temperature-sensitive origin produces a background of 10 "3 - 10 "4 . Thus, the use of the R6K ⁇ origin of replication very significantly reduces the background.
  • the second form of background is due to the cointegration of the conditional replication shuttle vector into the host cell DNA by undesired homologous recombination.
  • the third form of background is due to the conditional replication shuttle vector integrating into either the IOBCV or more likely, the host cell DNA by random recombination. The latter two factors are less significant than the first, which has been overcome by the use of the R6K ⁇ origin of replication as disclosed herein.
  • One such protocol can include:
  • Preferably two separate vials are electroporated for each BAC strain.
  • the Ori R6K ⁇ conditional replication shuttle vector can also carry a marker cassette containing a myc tag in all three reading frames followed by a stop codon, and/or an IRES/EGFP/polyA gene for creation of a fusion transcript expressing enhanced green fluorescent protein from an internal ribosome entry site (IRES).
  • IRES/EGFP/polyA gene for creation of a fusion transcript expressing enhanced green fluorescent protein from an internal ribosome entry site (IRES).
  • IRES/EGFP/polyA gene for creation of a fusion transcript expressing enhanced green fluorescent protein from an internal ribosome entry site (IRES).
  • IRES/EGFP/polyA gene for creation of a fusion transcript expressing enhanced green fluorescent protein from an internal ribosome entry site (IRES).
  • IRES/EGFP/polyA gene for creation of a fusion transcript expressing enhanced green fluorescent protein from an internal ribosome entry site (IRES).
  • IRES/EGFP/polyA gene for creation of a
  • the counterselection marker used can be the SacB gene.
  • the SacB gene encodes levansucrase, an enzyme that converts sucrose to levan, which is toxic to the host cells [Frengen et al, Genomics 58:250-253 (1999)].
  • a more preferred strategy has been developed that allows for high throughput liquid modification and resolution of a BBPAC, (e.g., a BAC see Example 3).
  • This method can employ a BAC shuttle vector that has been adapted from the shuttle vectors described above. Again, this particular vector can be modified from PLD55, and can contain a R6K ⁇ DNA origin of replication. As indicated above, the vector containing a R6K ⁇ DNA origin of replication can only replicate in bacteria expressing the pir replication protein, but it cannot replicate in DH10B, the host for the BACs. Therefore, it will not persist on its own in the BAC strains.
  • the vector also encodes a recombination protein, such as recA, which is used to transiently allow homologous recombination in the otherwise recombination deficient bacterial cells.
  • a recombination protein such as recA
  • recA a recombination protein
  • the cointegrates can be achieved through homologous recombination of the selected nucleic acid sequence inserted in the A box of the shuttle vector with the nucleotide sequence of the BAC (see Example 3).
  • the shuttle vector is designed to contain a specific drug resistant gene, such as Ala, which provides ampicillin resistance.
  • the cointegrates thus can be selected by growing the cells in LB media supplemented with the cooesponding antibiotic, e.g., ampicillin.
  • the double Ampicillin/ Chloramphenicol resistant colonies should contain the homologously recombined plasmids.
  • the BAC shuttle vector is constructed to also contain the positive counterselection marker, e.g., a SacB gene, which is lost upon final resolution (see Figure 22).
  • the SacB gene product levansucrase, converts sucrose to levan, which is toxic to the host cells.
  • the SacB gene facilitates the selection of resolved BAC clones when the media contains sucrose since unresolved BAC cointegrants still retain the counter-selection Sac B gene and are therefore, selected against when grown in media containing sucrose.
  • IRESEGFP can also be introduced into the shuttle vector, which contains the ribosome entry site (IRES) and expresses enhanced green fluorescent protein. Analyses of the expression of EGFP gene in transgenic mice, for example, allows the individual gene expression pattern to be observed.
  • IRESEGFP ribosome entry site
  • Ascl and Smal sites are also included in the shuttle vector preceding the marker gene. These two sites allow a selected nucleic acid sequence to be readily inserted in the A box of the shuttle vector allowing the preparation of the shuttle vector for directional cloning, see Example 3, with very little background due to failure of the recircularization of the vector.
  • a selected nucleic acid sequence, i.e., a homology region, from a gene of interest contained by an IOBCV can be amplified by PCR using one or more specific primers.
  • the amplified PCR product can then be placed into a conditional replication shuttle vector as indicated in Figure 20 in the "A box".
  • the first step in BAC modification is to design oligos for amplification of an approximately 300-500 basepair segment of the BAC.
  • Example 3' UTR region of the gene of interest was used for the preparation of the selected nucleic acid sequence to be placed in the A box.
  • the oligonucleotides can be designed to include an Ascl site at the 5' end of the amplified fragment because the shuttle vector (SV) is designed for very highly efficient and directional cloning of the "A box” fragments into an Ascl/Smal cleaved shuttle vector.
  • the PCR amplification of the "A box” can be done from any genomic DNA that is contained by an IOBCV (e.g., a BAC), including the DNA from C57BL/6J mice exemplified in Example 3 below. This ensures that the "A box” is isogenic with the BAC DNA (e.g. , from the BAC library RPCI 23 used in Example 3 below).
  • Cointegrates are selected after homologous recombination: As in the methods described above, each shuttle vector is transformed into an individual BAC containing strain. Homologous recombination can then occur between the shuttle vector and BAC. However, since the shuttle vector contains the R6K ⁇ origin, it cannot replicate in the BAC host cells, DH10B. Therefore, the selection for both the chloramphenicol marker on the BAC and the ampicillin marker on the shuttle vector yields only those colonies in which the cointegrates have been produced (other than the small background discussed above). The advantage of the R6K ⁇ is that it allows a dramatical improvement in the efficiency of the BAC modification procedure. Furthermore, the entire selection process can be done in a liquid culture simply by serial dilution (see Example 3).
  • the first step in either modification procedure occurs by homologous recombination through the approximately 500 basepair homologous "A box", discussed above to produce the cointegrate carrying both the marker and the shuttle vector sequences within the BAC.
  • the cells are grown in ampicillin and chloramphenicol to select for both the shuttle vector and the BAC.
  • the R6K ⁇ origin of replication in the shuttle vector cannot operate in the BAC strain and the shuttle vector plasmid cannot persist on its own.
  • the only way to obtain stable antibiotic resistance to both ampicillin and chloramphenicol is for the shuttle vector to integrate into the BAC or the host chromosome.
  • the cointegration can be upwards of 70% efficient with respect to the desired product and all of the selections could be transferred to liquid culture. This allows this step to occur in 96 well plates (or larger) so that concurrent modification of a large number of constructs can be achieved.
  • the next step in the first procedure is to express flp recombinase within the cointegrate containing cells to excise the shuttle vector sequences (see Figure 22).
  • the flp recombinase works via the "fit" sites surrounding the shuttle vector and it is highly efficient for excision [Hoang et al, Gene 212:77-86 (1998)].
  • a powerful negative selection of sucrose can be used relying on the SacB gene product.
  • An alternative protocol employs an integration resolution methodology, as the RecA activity is further exploited to allow an additional recombination step to complete the resolution of the modified IOBCV (e.g., a BAC).
  • the shuttle vector is constructed to contain two homologous sequences that are homologous to each other, but not homologous to the BAC.
  • Example 3 a second copy of IRESEGFP is used (see Figures 23 and 24). The first homologous recombination event occurs through the homologous nucleic acid of the "A box" to form the shuttle-vector-BAC cointegrate, whereas the second homologous recombination event serves to resolve out the vector sequence.
  • Example 3 the IRESEGFP sequence is much larger than the A box homology sequence that is used for the cointegration, and therefore, the resolution step occurs with much greater frequency through the IRESEGFP sequences (see Example 3).
  • the two homologous sequences that are homologous to each other, but not homologous to the BAC be longer than the selected nucleic acid sequence in the A box.
  • Bacterial based artificial chromosomes such as Bacterial artificial chromosomes (BACs) and P-l derived artificial chromosomes (PACs), are circular bacterial plasmids that may propogate as large as 300kb of exogenous genomic DNA [Shizuya et al, PNAS, 89:8794-8797, (1992); Sicilnou et al, Nature Genet., 6:84-90 (1994)].
  • the average size of the insert is 130-150 kb.
  • 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.
  • BBPACs 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 131kb 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. Materials and Methods 1. Isolation and initial mapping of BACs
  • 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) 2.
  • Solution II 0.2N NaOH, 1% SDS (0.4 g NaOH, 45 ml ddH20,
  • Solution III 5M KOAc (60ml), glacial acetic acid (11.5ml), H20 (28.5 ml).
  • Protocol 1). Inoculate each BAC containing bacterial to 50ml LB containing 12.5 ug/ml chloramphenicol. Grow overnight in 37°C.
  • 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).
  • 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 ohgonucleotides 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 ohgonucleotides 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).
  • Each sequencing reaction will result in up to a 500bp sequence. Sequence more than one BAC for a given primer to compare the sequences.
  • the main purpose for sequencing is to design a 20 bp PCR primer, which is about 500 bp away from the sequencing oligo (which usually is the other PCR primer), to enable PCR amplification of this genomic fragment and to clone it into the building vector. Therefore, as long as a 20bp sequence can be identified which is at the appropriate position, and which is the same in several independent sequencing reactions, the goal is achieved.
  • the quality of the DNA sequence in between is not very critical.
  • a two vector system is designed to construct the shuttle vector for BAC modification ( Figure 1).
  • 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 pSVl .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 (1 lkb), 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 Building Vectors
  • 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.
  • EGFPl (Fig. 10) This vector is designed to introduce the brighter version of the green fluorescent protein, EGFPl (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.
  • pBVIRES.EGFPl (Fig. 11) This vector is used to introduce EGFPl 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.
  • B Temperature sensitive, recombination inducing shuttle vector (pSVl.RecA) (Fig.
  • This plasmid vector was modified from the pMBO96 vector originally constructed by O'Connor et al (Science, 1989, Vol 244, pp.1307-1312).
  • the pMBO96 vector was a gift from Dr. Michael O'Connor.
  • the original vector carries tetracycline resistance, and contains a pSClOl temperature sensitive origin of replication, which allows the plasmid to replicate at 30°C but it will cease replication and is lost at 43°C.
  • the E. coli RecA gene was amplified by PCR and sub-cloned into the Bam HI site, to create the pSVl.RecA vector.
  • 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
  • the following plates Prior to cloning the recombination cassette into the shuttle vector, the following plates are usually prepared: the tetracycline (lOug/ml) LB agar plates and the tetracycline(10ug/ml) + chloramphenicol (12.5 ug/ml) LB agar plates. Plates are made according to standard protocol [Sambrook et al, (1989) supra].
  • pSVl .RecA and building vector midi-prep DNA by the alkaline lysis method ( see above).
  • Qiagen columns can also be used to obtain high purity DNA, though yield is usually low. This is due to the low copy number of the pSVl plasmid.
  • the culture should be grown at 30 °C in LB + tetracycline ( lOug/ml).
  • the final midi-prep DNA is usually dissolved in 200 ul TE or ddH 2 O.
  • Sal I is inactivated by heating to 65 °C for 15 minutes.
  • 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.
  • 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 pS V 1.vector, 100-200ng insert, 2ul 1 OX ligation buffer (Boehringer-
  • 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. Pick colonies and do colony hybridization according to standard protocols
  • 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 (1000X) 10 mg/ml in 50% ethanol, wrapped in aluminum foil and stored in -20°C for up to one month.
  • Chloramphenicol stock solution (1000X) 12.5 mg/ml, dissolved in ethanol (>50%), stored in -20 °C.
  • 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.
  • Chloramphenicol (12.5 mg/ml ) 0.5 ml 1 ml Pour the plates and leave the plates outside overnight and then store at 4 °C. There is no need to avoid the light.
  • a chemical method is used to prepare competent cells from BAC containing bacteria host (Inoue et al, Gene 96, p23-28, 1990).
  • TB media (lOmM Pipes, 55mM MnCl 2 , 15mM CaCl 2 and 250mM KC1), all the components except for MnCl 2 are mixed and the pH is adjusted to 6.7 with KOH. Then, MnCl 2 was dissolved, the solution was sterilized by filtration through a 0.45u filter unit and stored at 4°C. All salts were added as solids.
  • the cell pellet was gently resuspend in 4 ml of TB supplemented with 7% DMSO. Incubate on ice for lOmin, then dispense 0.5ml aliquot and immediately frozen by immersion into liquid nitrogen. The tubes are stored in -80 °C for further use.
  • the original BAC and the shuttle vector should be included in this analysis.
  • the reason to use the homology arms as Southern blot probes is that it will hybridize to two bands of appropriate size in the co-integrate BAC.
  • the original BAC and the shuttle vector should be included in this analysis.
  • Pulse field gel analyses should be done to confirm the modification event and to determine if there are any rearrangements in the modified BACs. Since there are two Not I site flanking the BAC insert (Research Genetics), digestion with Not I should reveal the size of the 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 the 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 the modified BAC with the original BAC will reveal any gross rearrangements in the modified BAC.
  • Probes used to hybridized to the PFGE blots include: insert specific probes (s.a. lacZ, PolyA, GFP and Neo) and whole BAC probe (to reveal all the digested bands from the BAC). Once the modified BACs are confirmed to have the specific targeted modification events and the lack of rearrangements, these BACs are ready to be used for the biological experiments, such as producing transgenic mice or transfecting cells.
  • the reservoir is put back with 10 ml of injection buffer in it. Now start collecting 0.5ml fraction with a 24 well plate. Generally about 12 fractions are collected (or until the blue dye is almost at the bottom of the column).
  • the bands should be visible after ethidium bromide staining.
  • a Southern blot is performed in order to choose the fractions with highest yield, and the least degradations.
  • 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 rec A 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 fusaric acid plates selects for the loss of tetracycline resistance, i.e., counterselecting 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 rec A 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).
  • 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)].
  • T e pSVl.RecA temperature sensitive shuttle vector containing the recombination cassette was transformed into the DH10 E.coli strain containing the BAC169 and selected by growth at either 30°C or 43 °C on plates containing chloramphenicol and tetracycline.
  • the co-integrates are then resolved as described above by growing the cells first on chloramphenicol plates at 43°C and then on chloramphenicol and fusaric acid plates at 37°C. Fusaric acid provides a strong counterselection against bacteria containing the tetracycline resistance gene. Indeed, 200 colonies picked from these plates were all tet sensitive, indicating the stringency of the selection. Duplicated colonies growing on the chloramphenicol plates were used for colony hybridization with the pgkpoly A probe. Eight out of 200 colonies were positive (4 %). Southern blot analyses using either homology at Al or Bl as the probe showed that all these clones contained correctly resolved BACs (Fig. 3C and 3D).
  • BACs Three BACs (lanes 4,5 and 8) also contained wild type bands, which may represent either contamination from other clones, or a BAC containing two copies of co-integrates that resolved through two different homologous regions.
  • the next step in our analysis was extensive mapping of the modified BACs to determine whether any unexpected deletions or insertions were generated during the modification procedure.
  • 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 (pgkpoly A) 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 site (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 BAC 169.
  • 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 in all three BACs. Additional fingerprinting of all eight modified BACs with Hindlll, EcoRI and Avrll digests showed that no detectable reaoangements 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. In this case, 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.
  • an appropriate injection buffer e.g., 100 mM NaCl, 10 mM Tris.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 ethidium bromide staining.
  • the SEPHAROSE CL-4B column could also efficiently separate the degraded DNA (in this case in fractions 3-6) from the pure linear DNA (fractions 7-9) (Fig.5A).
  • Fraction 8 contained 3 ⁇ g/ml DNA and was used directly for pronuclear injection.
  • Pronuclear injection into the fertilized C57BL/6 mouse zygote is performed according to a standard protocol [Hogan et al, in Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, New York, 1986)]. Two different concentrations of fraction 8 BAC DNA (obtained as described above) were used: 3 ⁇ g/ml and 0.6 ⁇ g/ml. No newborns were obtained with the high concentration DNA, suggesting that the high concentrations may be toxic to the zygote. However, with the lower concentration of pure linear DNA, 15 newborn mice were obtained and two of them (13%), Y7 and Y9, contained the lacZ marker gene as demonstrated on a Southern blot (Fig. 5B).
  • the intensity of the bands allows an estimate of 2-3 transgene copies for Y7 and one copy for Y9.
  • the presence of both ends of the BAC ends was assayed for in the transgenic mice. Since both BAC ends contain some vector sequence, PCR primers specific to the vector sequence were generated and used to amplify the transgenic DNA. The amplified products were then probed with a third labeled oligonucleotide probe within the amplified region. As shown in Fig. 5C and Fig. 5D: Y3, Y7 and Y9 have both ends present, while the negative controls do not.
  • Y7 and Y9 also have the lacZ gene, they are likely to contain intact BAC transgenes. For Y3, whereas it has both ends it does not contain the lacZ gene. This may be due to either a rearrangement or fragmentation during the injection prior to integration.
  • 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 littermates (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 O ⁇ 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.
  • loss-of- function phenotypes have played a central role in the discovery of complex morphogenetic pathways in a variety of organisms.
  • the seminal loss-of-function screens for genes affecting cell cycle traverse in yeast [Hartwell et al, Science 183(120):46-51 (1974)] and for mutations affecting early D. melanogaster development [Nusslein-Volhard and Wieschaus, Nature 287:795-801 (1980)] have provided the basis for our current understanding of cell division and of embryonic patterning.
  • loss-of- function genetics would not yield all the genes in the pathway under study, and alternative strategies for genetic analysis were therefore devised.
  • Granule cell precursors are specified during embryogenesis [Alder et al, Neuron 17:389-399 (1996)], forming a secondary proliferative zone called the external granular layer (EGL) [Miale and Sidman, Exp. Neurol 4:277-296 (1961); Fujita et al, J. Comp. Neurol. 128:191-208 (1996)]. Most mature granule cells arise in a period of rapid cell division that extends from birth until the acquisition of full motor function [Fujita et al, J. Comp. Neurol. 128:191-208 (1996); Airman, J. Comp. Neurol 136:269-294 (1969)] .
  • the Targeting vector contained a 3.7 kb 5' arm and a 6 kb 3' arm in a pKSNT vector [Tybulewicz et al, Cell 65:1153-1163 (1991)].
  • ES cell selections were performed at The Rockefeller University Gene Targeting Facility. Initial typing was done by Southern blot using a 500 bp pair probe from the 5' region (Fig. 3a) yielding a 15kb wildtype allele and 11.5 targeted allele upon digestion with BamHl.
  • transgenic mice Generation of the transgenic mice.
  • EGFPl marker gene internal ribosome entry site followed by a nucleic acid encoding an enhanced green fluorescent protein
  • the transgenic founders were in C57BL6/CBA Fl background and were backcrossed to C57BL6 in successive generations. Fl, F2 and F3 offsprings from this backcross were used for analyses.
  • ILPA were digested with EcoRI and Hz ' wdlll and separated on 1% agarose gel.
  • a Southern blot was prepared from the gel and was probed separately with the 1.6kb Xbal-Hindlll genomic DNA probe and the entire 131kb BAC169 probe.
  • Measurement of IGL area and granule cell density. P20 to P22 cerebella were taken and mounted in TISSUETEK mounting media for frozen sections, the IGL area was measured from a digitized image of a cresyl violet stained lO ⁇ m sections and the MacMeasure (NTH image) image analysis program. To minimize the variations due to weight differences, mice were weight-matched to be within 0.5 grams of the average weight.
  • the granule cell density was counted using a grid measuring 900 ⁇ m 2 that was randomly placed in the middle region of lobule V. For each cerebellum, six sections were counted and the average density was calculated. The statistical significance was calculated using a t-test.
  • Granule cell proliferation assay P8 cerebellar granule cells from the E6 line were prepared and cultured as re-aggregates as described [Gao et al, Neuron 6:705-714 (1991)]. At least six wells of 500,000 cells per well per genotype were used in each assay. In each well, 1.5 ⁇ Ci 3 H-thymidine (NEN) was added and the cells were incubated at 37°C for 22hrs. The cells were washed in CMF-PBS, lysed by adding 4 X volume of H 2 0 and the DNA were precipitated using 10% TCA. Incorporation of 3 H-thymidine was determined by scintillation counting of the precipitated material.
  • Cerebella from weight-matched P20-P22 Dl, E6 and Y7 mice were dissected out, lightly stained with cresyl violet and directly inspected for foliation pattern under a dissection microscope. Analysis of the skin phenotype. Animals were sacrificed and skin from the back near the midline was taken and fixed overnight in 75% Ethanol and 25% Acetic Acid. The tissue was dehydrated and embedded in paraffin. Tangential lO ⁇ m sections were taken for cresyl violet and anti-phospho H3 immunohistological staining.
  • Ru49 in vivo by gene targeting [M.R. Capecchi, Science 244:1288-1292 (1989)] and bacterial artificial chromosome (BAC) mediated transgenesis [Example 1 above, Yang et al, Nat. Biotech. 15:859-865 (1997)] is presented below. While no obvious phenotype was observed in Ru49 null mutant mice, increased Ru49 expression in vivo results in an increase in granule cell proliferative capacity and an increase in granule cell number. The formation of intralobular fissures is elevated in the Ru49 BAC transgenic mice, revealing an important role for this factor in cerebellar morphogenesis.
  • Ru49 loss-of-function genetic analysis To test the role of Ru49 in vivo, mice were prepared with a targeted loss-of-function mutation at the Ru49 locus. As shown in Fig. ⁇ 3a, the gene targeting vector employed for this work resulted in replacement of the entire first coding exon and half of the second exon of the Ru49 gene with the neo gene [Southern and Berg, J. Mol. Appl Genet 4:327-341 (1982)]. This results in deletion of both the Ru49 activation domain [Chowdhury et al, Mech. Dev. 39:129- 141 (1992)] and the LeR or SCAN box domains [Pengue et al, Nucleic Acids Res.
  • Ru49 ⁇ mice are born in a Mendelian ratio, they are fertile and display no apparent morphological or behavioral abnormalities.
  • both the size and morphology of the cerebella o ⁇ Ru49 '/' mice were measured relative to their Ru49 +/ ⁇ and wild type httermates (Fig. 13/3). No statistically significant difference in the size or morphology of the cerebellum was evident in these animals (Figs. 13c and 13d).
  • BAC169tEGFP a modified BAC construct
  • IRES/EGFP marker gene Figs.14a and 14b
  • IRES/EGFP marker gene Figs.14a and 14b
  • the fusion transcript translates two proteins, epitope-tagged Ru49 and EGFPl.
  • IRES internal ribosome entry site
  • ILPA transgenic line was used [Yang et al, Nat. Biotech. 15:859-865 (1997), Example 1 above].
  • the BAC169.ILPA BAC contains identical sequences outside of the Ru49 gene, but does not express the Ru49 protein (Fig. 14c).
  • detailed restriction mapping of the original BAC 169 and the two derivatives, BAC169.ILPA and BAC169tEGFP, were performed using restriction digests with Notl, Pmel, Xbal, Xhol, EcoRI and H dIII.
  • EcoRl and Hindll digests in Fig.l4 Southern blots using the 1.6kb Xba-Hindl ⁇ l probe from the Ru49 locus reveal the correct targeted D ⁇ A fragments for both BAC169.t ⁇ GFP and BAC169.ILPA.
  • Four transgenic mouse lines carrying BAC169tEGFP were produced (Fig. 15). Copy number analysis shows that the A4 line contains fourteen copies, the Dl and the E6 line six copies each, and the B8 line one copy. Lines Dl and E6 were chosen for further analysis because the BAC transgene copy number in these strains was comparable to that in the control Y7 line (four copies).
  • Northern blot analysis showed that the Dl and E6 transgenic cerebella expressed a 3.8 kb fusion rnRNA containing both the Ru49 and the IRES.
  • EGFPl sequences, in addition to the wildtype 2.2 kb Ru49 rnRNA (Figs.
  • the Dl line expresses approximately 4 fold and the E6 line 5 times the wildtype level o ⁇ Ru49 transcript.
  • the BAC169.ILPA line Fig. 5g
  • the BAC169tEGFP lines expressed the EGFP marker proteins at a higher level in the EGL than in the IGL of the developing cerebellum (Figs. 15e and 15f), as expected from the pattern of expression of the endogenous Ru49 gene [Yang et al, Development 122:555-566 (1996)].
  • the transgenic animals also produce epitope tagged Ru49 protein of the expected size in the cerebellum (Fig. 15d). Correct expression was also seen in the dentate gyrus, olfactory bulb, thymus, testis and skin; no ectopic expression was observed.
  • the Dl, E6 and Y7 transgenic mice are fertile, of normal weight and longevity, and have no apparent motor or behavioral abnormalities.
  • the cerebellum of P10 to P20 transgenic mice are consistently larger than those oftheir wildtype httermates, as shown in whole mount (Fig. 16a) or in midsagittal section of the cerebellar vermus (Figs. 16b and 16c). Since Ru49 overexpression is restricted to the granule cell lineage, the granule cell number was estimated by measuring the IGL area and granule cell density in vermal sagittal sections from the Dl and E6 cerebella.
  • Ru49 is important for formation of intralobular fissures in the cerebellum. Granule cell proliferation is postulated to play an important role in the formation of the cerebellar fissures and lobules [Mares and Lodin, Brain Res. 23:343-352 (1970)].
  • the Ru49 over-expressing mice also displayed an acceleration in the formation of intralobular fissures.
  • ICF intraculminate fissure
  • Ru49 transgenes in mouse skin is evident from the marker gene expression patterns in the BAC 169. ILPA and BAC169.tEGFP transgenic mice relative to their wild-type httermates. Examination of the lacZ expression in the Y7 line revealed cells highly expressing lacZ in the hair follicles and sebaceous glands, as well as faint general staining in other cells of the epidermis (Figs. 19b and 19c). EGFP expression in the skin of BAC169.tEGFP transgenic mice was detected immunocytochemically, confirming the lacZ expression results and demonstrating highest levels of expression in the hair follicles and sebaceous glands (Figs.
  • the epidermis itself appears to be relatively unaffected in the transgenic mice. Since the expression of the lacZ marker gene is concentrated in these abnormal hair follicles and in the sebaceous gland these data suggest that elevated Ru49 protein acts intrinsically in these cells to perturb hair follicle development.
  • Shh can induce proliferation of cerebellar granule cell precursors in vitro and in situ, and production of Shh blocking antibodies in the developing cerebellum strongly inhibits the generation of granule cells in vivo [Wechsler-Reya and Scott, Neuron 22:103-114 (1999)]. These results establish Shh as a major mitogen for granule cell precursors.
  • the effects ofRu49 on granule cell production suggest that Ru49 could participate in the response of granule cells to Shh. The simple idea that Ru49 can directly regulate the response to Shh by modulating tc expression levels appears to be incorrect, since the expression of ptc rnRNA is not altered in Ru49 overexpressing animals.
  • Ru49 expression or function is regulated by the Shh pathway, and that its role in the cell is to increase the proliferative response through an intrinsic cellular pathway.
  • Ru49 is a target for the Shh pathway, and the genes regulated by Ru49 play an important role in regulating traversal through the cell cycle.
  • the present results demonstrate a direct relationship between postnatal granule cell proliferative capacity and the formation of intralobular cerebellar fissures. Since the fissures formed as a consequence of increased Ru49 gene dosage also occur naturally as heritable morphological differences between inbred mouse strains [Tnouye and Oda, J. Comp. Neurol 190:357-362 (1980); Neumann et al, Brain Res. 524:85- 89 (1990)], increased Ru49 expression does not alter patterning of the cerebellum.
  • Ru49 BAC transgenic animals A role for Ru49 in hair follicle development and epidermal cell proliferation: The present investigation of the skin in Ru49 BAC transgenic animals was prompted by the observation that both the lacZ and EGFP marker genes were expressed in the skin, which caused us to reevaluate the initial in situ hybridization analyses and to confirm that this is a site of expression for the endogenous Ru49 gene.
  • Thyroid hormone has been shown to influence the formation of the same cerebellar intralobular fissures affected by increased Ru49 gene dosage [Lauder et al, Brain Res. 76:33-40 (1974)], suggesting that Ru49 and hairless could function in the same hormonally responsive genetic pathway to regulate proliferation in the cerebellum and skin. Since the proliferation of precursor cells in vivo is probably controlled by the integration of a complex set of genetic and environmental factors, it is also possible that Ru49 operates downstream from both Shh and thyroid hormone in an intrinsic cellular pathway.
  • BAC transgenic mice as tools for genetic research.
  • both a traditional loss-of-function mouse mutant as well as BAC transgenic mouse lines with increased Ru49 gene dosage were generated.
  • the loss-of-function mutant mice have no obvious phenotype in the cerebellum or other tissues.
  • the Ru49 BAC transgenic lines expressing increased levels of the transcription factor display several specific phenotypes that document a role for Ru49 in proliferation of granule cell precursors in the developing cerebellum, and progenitor cell division during early postnatal development of the hair follicles. From these results, it can be concluded that both that Ru49 function is redundant in the mouse, and that important insights into its function can be obtained from increased gene dosage.
  • transgenic mice carrying BACs from the mouse Clock locus both rescue the long period and loss-of-rhythm phenotypes of the original Clock mutation, and shorten the circadian period on the wild type background [Antoch et al, Cell 89:655-667 (1989)].
  • BACs often contain all the information necessary for correct copy number-dependent and position-independent transcription in transgenic mice
  • cDNA in eukaryotic cells and transgenic mice has been widely used for the study of gene function and regulation.
  • the cDNA itself is often missing important elements for regulation of gene activity, such as high-level, tissue-specific, and integration site-independent expression of the transgene. Those elements such as enhancers, locus control regions (LCR), and insulators, may reside at a large distance (>50kb) from the gene itself.
  • LCR locus control regions
  • insulators may reside at a large distance (>50kb) from the gene itself.
  • a intact genomic loci as a transgene will be essential for this expression.
  • Bacterial artificial chromosome (BACs) and P-l derived artificial chromosomes (PACs) have become a widespread and powerful resource in manipulating the large genomic DNA in E. coli.
  • BAC transgenic technology has been used for studying gene function and regulation, the efficiency for modifying and resolving these BACs can be improved.
  • a strategy has been developed that allows for liquid modification, and high throughput.
  • This method employs specific elements that are constructed in a BAC shuttle vector.
  • a R6K ⁇ DNA replication origin which requires the expression of the pir protein is included to allow selective reproduction/dilution of the shuttle vector depending on the strain of bacteria containing the shuttle vector.
  • a gene encoding a recombination protein e.g., rec A, is employed to transiently allow homologous recombination.
  • the shuttle vector also comprises a specific drug resistant gene, e.g. , Ala, (the BAC contains a chloramphenicol-resistant gene).
  • the shuttle vector includes a positive counterselection marker, e.g., the SacB gene.
  • the shuttle vector also comprises a marker gene, e.g., IRESEGFP that is adjacent to the A box, (homology region) thereby enabling the detection of the gene product of the gene of interest comprised in the BAC when it is expressed.
  • Fifth, Ascl and Smal sites are introduced into the shuttle vector surrounding the A box.
  • the shuttle vector also comprises two FRT sites (see Figure 20).
  • the shuttle vector has two copies of the IRES EGFP marker that bracket the desired insert (see Figure 23) rather than the two FRT sites (see Figure 23).
  • the replication origin for this vector allows growth at a high copy number in strains containing the pir gene, but it cannot replicate in DH10B, the host for the BACs [Metcalf et al, Plasmid 35:1-13 (1996)]. This is advantageous both because it is very simple to obtain large amounts of DNA for cloning into this vector, and because the plasmid cannot persist on its own in the BAC strain.
  • the Ascl and Smal sites and corresponding restriction enzymes are used because they allow the preparation of the shuttle vector for directional cloning of the "A box" with very little background due to failure of the recircularization of the vector. Following ligation, approximately 50% of the colonies plated contained the PCR amplified insert when this vector was used. Finally, the SacB gene is added to the vector because it is a powerful negative selectable marker for use in subsequent steps of the modification protocol.
  • a protocol for cloning of the shuttle vectors for each BAC is as follows:
  • Homology regions from a gene of interest from C57bl/6J genomic DNA is amplified by PCR using primers to the 3'UTRs of the gene of interest (using a 5 'primer to incorporate the ASCI site).
  • the products are digested overnight with Ascl, and the digested fragments are purified by gel electrophoresis in low melting point agarose (one gel per week for at least twenty amplified fragments).
  • the agarose is melted and the digested shuttle vector (lOOng) from step (1) above, are ligated with the purified fragments (25ng) from step (2), transformed and plated on LB amp plates. The ligation occurs between the "A" box of the shuttle vector and the PCR fragments from the genomic DNA.
  • a few colonies (e.g., 4) per ligation are picked individually and tested for correct insertion by PCR using a 5' end primer spanning from the shuttle vector to the gene specific 3' end primer used to amplify the "A box".
  • DNA minipreps are then prepared for positive shuttle vectors for each gene for use in modification.
  • the shuttle vectors now contain the nucleic acid fragment in the "A" box that is to be inserted into the gene of interest of the BAC.
  • a protocol for preparation of the cointegrates is as follows: 1.
  • the PLD55-modified shuttle vector containing a selected nucleic acid sequence, i.e., an homology region, for the gene of interest which is contained by a BAC is transformed into BAC competent cells by electroporation. 40ul of competent cells containing the BAC are thawed on ice, and then mixed with 2 ⁇ l of
  • a Gene Pulser apparatus is used to carry out the electroporation.
  • the Gene Pulser apparatus is set at 25 ⁇ F, the voltage to 1.8KV and pulse controller to 200 ⁇ .
  • the transformed cells are select with 5 ml of LB supplemented with chloramphenicol (12.5 ⁇ g/ ⁇ l) and ampicillin (25 ⁇ g/ml), and then incubated at 37°C overnight.
  • the overnight culture is diluted 1 to 1000 and grown in 5 ml of LB with chloramphenicol (12.5 ⁇ g/ml) and ampicillin (50 ⁇ g/ml)at 37°C for about six hours.
  • This culture is diluted 1 to 5000 and grown in the same media at 37°C for about 4 to 5 hours. A series of dilutions are made, and they are placed on Amp plates, incubated at 37°C overnight.
  • the flp recombinase works via the "frt" sites surrounding the shuttle vector and it is highly efficient for excision (see Figure 20).
  • a protocol for this procedure is as follows: 1. Each individual bacterial colony containing the cointegrate is grown in 1 ml of
  • the cells are spun down at 3000 rpm and each sample is resuspended with 100 mM CaCl 2 .
  • the cells are transformed with a plasmid containing the AraBADflp, a kanamycin resistant gene and a temperature sensitive origin.
  • the cells are then grown on Kana/Chl plates at 30°C overnight to select for transformants containing the Flp recombinase plasmid.
  • the colonies (e.g., 3) picked up are placed in 3 ml of LB supplemented with Kanamycin and Chloramphenicol, and grown until an OD 600 of about 0.5 is observed. Flp recombinase is induced with arabinose for three hours.
  • DNA is prepared from a 10 ml culture (40 total), and the location of the gene within the BAC is mapped by digestion with Ascl and Notl. Pulsed field gel electrophoresis is used to detect the introduced Ascl site relative to the ends of the genomic DNA insert (Not 1 sites).
  • Preferred BAC construct DNA is selected, and when desired, prepared for transgenic injection.
  • An alternative method for this step of the modification utilizes the same homologous recombination event to form the cointegrate, but a second homologous recombination event to resolve out the vector sequences.
  • the shuttle vector has two copies of the IRES EGFP marker that bracket the desired insert (see Figure 23) rather than the two FRT sites.
  • this shuttle vector integrates into the BAC, it produces a cointegrate that is depicted in Figure 24.
  • the cointegrate Since the duplicated EGFP is not homologous to the BAC, the cointegrate always forms through the "A box", again with close to 100% efficiency. However, the two copies of the EGFP now flank the shuttle vector and these can be used for homologous recombination to resolve the cointegrate to get rid of the shuttle vector. Since the EGFP is much larger than the A box homology region that was used for cointegration, the resolution step occurs with much greater frequency through the EGFP sequences. If the negative selection for SacB is sufficiently strong, the percentage of correctly resolved modified BACs should be comparable to those seen with the Flp/FRT system. This system has been tested yielding 80% correct cointegration events and 100% correct resolution of those cointegrated plasmids.
  • Each colony of cointegrate is picked up from the Amp plates. Each colony is then inoculated with 5 ml of LB supplemented with chloramphenicol(12.5 ⁇ g/ml) and
  • the culture is next diluted 1 to 5000 and then plated on the agar plate with chloramphenicol(12.5 ⁇ g/m ⁇ ) and 6% sucrose, and incubated at 37°C overnight.

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Abstract

L'invention concerne un procédé efficace à haut débit visant à modifier des gènes dans une cellule hôte présentant un déficit de recombinaison. Ces modifications comprennent la production de segments d'insertion, de délétions, de substitutions, et/ou de mutations ponctuelles à n'importe quel site choisi du vecteur de clonage à origine indépendante. Le gène modifié est contenu dans un vecteur de clonage à origine indépendante qui est utilisé pour introduire un gène hétérologue modifié dans une cellule. Un tel vecteur modifié peut être utilisé dans la production d'un animal transgénique ayant reçu des cellules germinales, ou dans des protocoles de ciblage de gène mis en oeuvre dans des cellules eucaryotes.
PCT/US2000/019926 1999-07-20 2000-07-20 Recombinaison homologue conditionnelle de grands segments d'insertion de vecteur genomiques WO2001005962A1 (fr)

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WO2009114185A2 (fr) 2008-03-12 2009-09-17 The Rockefeller University Procédés et compositions pour profil translationnel et phénotypage moléculaire
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
US9096909B2 (en) 2009-07-23 2015-08-04 Chromatin, Inc. Sorghum centromere sequences and minichromosomes

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CN111471646B (zh) * 2020-04-20 2022-07-05 安徽中盛溯源生物科技有限公司 利用非抗生素筛选的游离型质粒载体制备iPSC的方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
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
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
US9096909B2 (en) 2009-07-23 2015-08-04 Chromatin, Inc. Sorghum centromere sequences and minichromosomes

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