International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 116 - MUTATION  IN A CRYPTIC 3’SPLICE SITE OF THE NEO GENE IN U3NEO GENE TRAP VECTOR DOES NOT DECREASE THE NUMBER OF INTRON INSERTIONS.

Dr. Anna Osipovich
Vanderbilt University
AA4206, MCN, Vanderbilt University School of Medicine,
1161 21st Avenue South
Nashville
37232
USA

Co-Authors: White EK, Ruley HE
Institutions: Department of Microbiology and Immunology, Vanderbilt University School of Medicine

Our laboratory has developed a strategy for large-scale insertional mutagenesis in mice called “tagged sequence mutagenesis”. This process uses a retrovirus shuttle vector U3Neo to clone and sequence  regions of cellular genes disrupted by virus integration. In analyzed ES cell clones nearly half of provirus insertions into characterized genes were in introns. In most cases, Neo expression was possible because fusion transcripts utilized a cryptic 3’SS located in the Neo coding region. To decrease the number of intron insertions and increase the efficacy of U3Neo shuttle vector we introduced substitution(A-T) mutation into the Neo cryptic 3’SS. A library of ES cells with inserted mutant U3Neo2 vector has been created and analyzed. Unexpectedly, U3Neo2  also inserted in introns  with the same frequency as U3Neo, but now the cryptic SS was located in the adjacent  intron. These results clarify mechanisms that allow expression of U3 gene trap vectors and provide evidence for the exon definition model of splice site selection. While cryptic SS's  located in introns  of cellular genes are not normally recognized by  the splicing machinery, they can be used efficiently if the context is altered by the insertion of  a Neo half terminal exon. Activation of the cryptic 3' SS is required for the expression of transcripts that contain the Neo coding sequence within a functional 3' terminal exon.


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