US 6,136,566 AGranted

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

AssigneeLexicon Graphics Incorporated

InventorsArthur Sands, Glenn Friedrich, Brian Zambrowicz, Allan Bradley

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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.

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 site operatively positioned 5' to the initiation codon of said 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 end of said foreign exon and said splice donor site;
said vector not comprising a polyadenylation site operatively positioned 3' to the coding region of said foreign exon;
and
said vector not comprising a promoter element operatively positioned 5' to the coding region of said foreign exon;
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 first splice donor site operatively positioned 3' to said foreign exon;
an exon comprising a second splice donor site upstream from said promoter and a splice acceptor upstream from said second splice donor site;
and
said vector not comprising a transcription terminator or polyadenylation site operatively positioned relative to the coding region of said foreign exon;
and
said vector not comprising a splice acceptor site operatively positioned between said promoter element and the initiation codon of said foreign exon;
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.

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Abstract

Claims23