US 6,207,371 B1 · Indexed library of cells containing genomic modifications and methods of making and utilizing the same

Abstract

Methods and vectors (both DNA and retroviral) are provided for the construction of a Library of mutated cells. The Library will preferably contain mutations in essentially all genes present in the genome of the cells. The nature of the Library and the vectors allow for methods of screening for mutations in specific genes, and for gathering nucleotide sequence data from each mutated gene to provide a database of tagged gene sequences. Such a database provides a means to access the individual mutant cell clones contained in the Library. The invention includes the described Library, methods of making the same, and vectors used to construct the Library. Methods are also provided for accessing individual parts of the Library either by sequence or by pooling and screening. The invention also provides for the generation of non-human transgenic animals which are mutant for specific genes as isolated and generated from the cells of the Library.

Claims38

Dependent claims

A collection of cultured eucaryotic cells made by a process comprising:

a) treating a first group of cells with a first vector to stably integrate into the genome of said cells, said first vector mediates the splicing of a foreign exon internal to a cellular transcript and said first vector comprising:
a foreign exon;
a splice acceptor sequence operatively positioned 5′ to the foreign exon;
a splice donor site operatively positioned 3′ to said foreign exon;
and
a sequence comprising a nested set of stop codons in each of the three reading frames located between the 3′ end of said foreign exon and said splice donor site;
b) treating a second group of cells with a second vector to stably integrate into the genome of said cells, said second vector mediates the splicing of a foreign exon 5′ to an exon of a cellular transcript and said second vector comprising:
a foreign exon;
a promoter element operatively positioned 5′ to said foreign exon;
a splice donor site operatively positioned 3′ to said foreign exon;
and
a mutagenic foreign polynucleotide sequence positioned upstream to said promoter;
and
c) selecting for cells from the first group of cells and/or the second group of cells that express the products encoded by the foreign exon;
whereby in the method a collection of cultured eucaryotic cells is made.

12 more elements

A vector for inserting a foreign mutagenic polynucleotide sequence internal to an animal cell transcript, comprising:

a) a foreign exon;
b) a splice acceptor sequence operatively positioned 5′ to the foreign exon;
c) a splice donor site operatively positioned 3′ to said foreign exon;
d) a sequence comprising a nested set of stop codons in each of the three reading frames located between the 3′ end of said foreign exon and said splice donor site;
e) said vector not comprising a polyadenylation site operatively positioned 3′ to said foreign exon;
and
f) said vector not comprising a promoter element operatively positioned 5′ to the coding region of said foreign exon.

4 more elements

A genetically engineered vector comprising:

a) a 5′ gene trap cassette comprising in operable combination:
1) a splice acceptor;
2) a first exon sequence located 3′ to said splice acceptor, said first exon encoding a marker enabling the identification of a cell expressing said exon;
and
3) a polyadenylation sequence defining the 3′ end of said first exon;
and
b) a 3′ gene trap cassette located 3′ to said polyadenylation sequence and comprising in operable combination:
1) a promoter;
2) a second exon sequence located 3′ from and expressed by said promoter;
and
3) a splice donor sequence defining the 3′ region of the exon;
c) a mutagenic foreign polynucleotide sequence positioned downstream of said first exon and upstream of said promoter of said 3′ gene trap cassette;
and wherein said vector does not encode a promoter mediating the expression of said first exon, and wherein said vector does not encode a sequence that mediates the polyadenylation of an mRNA transcript encoded by said second exon sequence and expressed by said promoter.

10 more elements

Dependencies are inferred from the claim text.

Indexed library of cells containing genomic modifications and methods of making and utilizing the same

Life of this patent

Indexed library of cells containing genomic modifications and methods of making and utilizing the same

Life of this patent

Abstract

Claims38