Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-10
2003-01-07
McKane, Joseph K. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C435S471000, C435S481000, C435S483000, C435S488000, C435S091500, C435S091500
Reexamination Certificate
active
06503712
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to generation of genomic libraries in shuttle vectors and high throughput generation of vectors by homologous recombination for creating transgenic animals by homologous recombination.
BACKGROUND OF THE INVENTION
A particularly productive approach to understanding the function of a particular gene in animals involves the disruption of the gene's function by “targeted mutagenesis”. A common form of targeted mutagenesis is to generate “gene knockouts”. Typically, a gene knockout involves disrupting a gene in the germline of an animal at an early embryonic stage. (See, Thomas et al.,
Cell
, 51:503 (1987).) Once established in the germline, it is possible to determine the effect of the mutation on the animal in both the heterozygous and homozygous states by appropriate breeding of mice having the germline mutation.
The mouse knockout model system is very useful for functional genomic analysis of genes. The advantages of mouse models for the study of mammalian physiology, and testing of therapies for the treatment of human diseases, and developmental abnormalities have been extensively established.
Among the many examples of the use of knockout technology utilized to investigate gene function are U.S. Pat. Nos. 5,625,122 and 5,530,178 to Mak, T. which describe the production of mice having a disrupted gene encoding lymphocyte-specific tyrosine kinase p56
lck
and Lyt-2, respectively. Silva et al.,
Science
, 257:201 (1992) produced mice having a disrupted &agr;-Calcium Calmodulin kinase II gene (&agr;CaMKII gene) which resulted in animals having an abnormal fear response and aggressive behavior. (See, also, Chen et al.,
Science
, 266:291 [1994]). Wang et al.,
Science
, 269:1108 (1995) demonstrated that the disruption in mice of the C/EPB&agr; gene which encodes a basic leucine zipper transcription factor results in impaired energy homeostasis in the mutant animals. Knudsen et al.,
Science
, 270:960 (1995) demonstrated that disruption of the BAX gene in mice results in lymphoid hyperplasia and male germ cell death.
The most common approach to producing knockout animals involves the disruption of a target gene by inserting into the target gene (usually in embryonic stem cells), via homologous recombination, a DNA construct encoding a selectable marker gene flanked by DNA sequences homologous to part of the target gene. When properly designed, the DNA construct effectively integrates into and disrupts the targeted gene thereby preventing expression of an active gene product encoded by that gene.
Homologous recombination involves recombination between two genetic elements (either extrachromosomally, intrachromosomally, or between an extrachromosomal element and a chromosomal locus) via homologous DNA sequences, which results in the physical exchange of DNA between the genetic elements. Homologous recombination is not limited to mammalian cells but also occurs in bacterial cells, yeast cells, in the slime mold
Dictyostelium discoideum
and in other organisms. For a review of homologous recombination in mammalian cells, see Bollag et al.,
Ann. Rev. Genet
., 23:199-225 (1989) (incorporated herein by reference). For a review of homologous recombination in fungal cells, see Orr-Weaver et al.,
Microbiol. Reviews
, 49:33-58 (1985) incorporated herein by reference.
With the increasing awareness that animal, and particularly mouse mutations can provide such useful insights about the function of genes from humans, a great deal of interest is developing to systematically generate mutations within genes in mice that correspond to those genes which are being isolated and characterized as part of various genome initiatives such as the Human Genome Project. The problem with utilizing these procedures for large-scale mutagenesis experiments is that the technologies for generating transgenic animals and targeted mutations are currently very tedious, expensive, and labor intensive. The most tedious parts of making an animal knockout construct from a given cDNA is obtaining an appropriate genomic fragment and gene mapping. Once the genomic fragment is obtained and mapped, actual assembly of the targeting vector also is a tedious process depending upon availability of appropriate restriction sites.
Generally, the preparation of these constructs requires isolating genomic clones containing the region of interest, developing restriction maps, engineering restriction sites into the clones, and restriction digesting and ligating fragments to engineer the specific construct needed to produce the knockout. See, e.g., Mak, T. U.S. Pat. Nos. 5,625,122 and 5,530,178; Joyner et al.,
Nature
, 338:153-156 (1989); Thomas et al., supra; Silva et al., supra, Chen et al., supra; Wang et al., supra; and Knudsen et al., supra. This is a long and tedious process that can take several months to complete. Thus, in order to more rapidly and efficiently create model organisms with genomic modifications, there exists a need to develop high throughput methods for the production of targeting constructs which do not require identification of target genomic fragments by traditional means, their cloning, and subsequent restriction mapping and other complex molecular engineering steps.
SUMMARY OF THE INVENTION
The present invention, in preferred embodiments, provides methods of preparing a genomic library for use in producing knockout targeting vectors comprising preparing a size selected mouse genomic DNA; preparing a shuttle vector comprising inserting said genomic DNA into a yeast vector, wherein the vector comprises a first bacterial origin of replication; a first bacterial selection marker; a first yeast origin of replication; a first yeast selection marker; and a first mammalian selection marker; transforming bacterial host cells with said shuttle vector to amplify said genomic library; arraying said transformed host cells into pools of cloned cells comprising shuttle vectors comprising a genomic DNA fragment; a first yeast origin of replication; a first yeast selection marker; a first bacterial origin of replication; a first bacterial selection marker; and a first selection marker for integration into mammalian cells; wherein the cells in said pools comprise mouse genomic fragments of different size.
In specific embodiments, the genomic DNA is a library which comprises mouse genomic DNA fragments ranging from about 8 kb to about 14 kb.
More particularly, the mouse genomic DNA fragments are isolated from a mouse strain selected from the group consisting of 129svj, 129 Ola, 129sv, and C57BL/6. Of course, these are merely exemplary strains of mice and those of skill in the art will be aware that other mouse strains may be employed for generating the transgenic animals of the present invention. Likewise, while certain preferred embodiments are directed to the generation of transgenic mice, it should be understood that the present invention is equally applicable to generating transgenic animals of other species such as, for example, mammals including but not limited to rabbits, mice, rats, hamsters, goats, sheep, pigs, horses, cows, dogs, cats, as well as primates, such as, monkeys, apes, and baboons.
In specific embodiments, the genomic library, when transformed into the bacterial host cells with said shuttle vector generates between about 3×10
6
and 5×10
6
clones. This is an exemplary range and it is contemplated that those of skill in the art may prepare a genomic library that generates more or fewer clones. Thus the practice of the invention may generate about 1×10
6
clones, about 2×10
6
clones, about 3×10
6
clones, about 4×10
6
clones, about 5×10
6
clones, about 6×10
6
clones, about 7×10
6
clones, about 8×10
6
clones, about 9×10
6
clones, about 10×10
6
clones or more clones or indeed may generate less than 1×10
6
clones and still provide meaningful shuttle vectors that may be used in the context of the present invention. The host cells that are available for tran
Amgen Inc.
Friend Thomas
Marshall Gerstein & Borun
McKane Joseph K.
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