Enrichment of insertional mutants following retrovirus gene trap selection.
The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.
Chang
W
W
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Hubbard
S C
SC
Friedel
C
C
Ruley
H E
HE
D016475:3T3 Cells; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004171:Diploidy; D005822:Genetic Vectors; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D009844:Oligosaccharides / Q000032:analysis / Q000096:biosynthesis; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D016662:Virus Integration
D009844:Oligosaccharides; D004247:DNA; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D013937:Thymidine Kinase; D008358:Mannose