Methods and compositions for preparing a genomic library for knockout targeting vectors
First Claim
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1. A method of preparing a genomic library for use in producing knockout targeting vectors comprising:
- a) preparing genomic DNA fragments of pre-selected sizes wherein said genomic DNA comprises mouse genomic DNA fragments ranging from 8 kb to 14 kb;
b) preparing a shuttle vector comprising inserting said genomic DNA fragments into a yeast vector wherein said yeast vector is pYYL-1, which comprises;
i) a bacterial origin of replication;
ii) a bacterial selection marker;
iii) a yeast origin of replication;
iv) a yeast selection marker; and
v) a selectable marker for selection of mammalian cells;
c) introducing said vector into bacterial host cells to amplify said shuttle vectors in transformed bacterial host cells; and
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d) arraying said transformed bacterial host cells into pools wherein the bacterial host cells comprise said shuttle vectors and wherein said pools comprise genomic fragments of pre-selected sizes wherein said pYYL-1 vector comprises a 4.4 kb fragment, generated through a SphI restriction digestion of yeast-E. coli shuttle vector pGBT9, wherein said 4.4 kb fragment is annealed at the SphI sites to a first oligonucleotide containing a SP6 site and a second oligonucleotide containing a T7 site.
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Abstract
The present invention is directed to methods for producing gene targeting constructs by homologous recombination using mouse genomic libraries arrayed in yeast shuttle vectors. The invention is also directed to methods of using targeting constructs made by the methods to generate transgenic animals.
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Citations
24 Claims
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1. A method of preparing a genomic library for use in producing knockout targeting vectors comprising:
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a) preparing genomic DNA fragments of pre-selected sizes wherein said genomic DNA comprises mouse genomic DNA fragments ranging from 8 kb to 14 kb;
b) preparing a shuttle vector comprising inserting said genomic DNA fragments into a yeast vector wherein said yeast vector is pYYL-1, which comprises;
i) a bacterial origin of replication;
ii) a bacterial selection marker;
iii) a yeast origin of replication;
iv) a yeast selection marker; and
v) a selectable marker for selection of mammalian cells;
c) introducing said vector into bacterial host cells to amplify said shuttle vectors in transformed bacterial host cells; and
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d) arraying said transformed bacterial host cells into pools wherein the bacterial host cells comprise said shuttle vectors and wherein said pools comprise genomic fragments of pre-selected sizes wherein said pYYL-1 vector comprises a 4.4 kb fragment, generated through a SphI restriction digestion of yeast-E. coli shuttle vector pGBT9, wherein said 4.4 kb fragment is annealed at the SphI sites to a first oligonucleotide containing a SP6 site and a second oligonucleotide containing a T7 site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of preparing a genomic library comprising:
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a) preparing size selected genomic DNA ranging from 8 kb to 14 kb in size;
b) preparing a shuttle vector comprising said genomic DNA and a the pYYL-1 yeast vector, wherein said pYYL-1 vector comprises a 4.4. kb fragment, generated through a SphI restriction digestion of yeast-E. coli shuttle vector pGBT9, wherein said 4.4 kb fragment is annealed at the SphI sites to a first oligonucleotide containing a SP6 site and a second oligonucleotide containing a T7 site;
c) amplifying said shuttle vector in transformed bacterial host cells; and
d) arraying said transformed bacterial host cells into pools wherein the bacterial host cells comprise said shuttle vectors and wherein said pools comprise a library of genomic fragments of pre-selected sizes. - View Dependent Claims (22, 23, 24)
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Specification