title: Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene.
abstract: BACKGROUND
  Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission.
  
  
  RESULTS
  Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A) site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression.
  
  
  CONCLUSIONS
  Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.
authors.affiliation: Department of Microbiology and Immunology, Room AA4210 MCN, Vanderbilt University School of Medicine, 1161 21st Ave South, Nashville, TN, 37232-2363, USA. mroshon@carolinas.org
authors.forename: Michael
authors.initials: M
authors.lastname: Roshon
authors.forename: James V
authors.initials: JV
authors.lastname: DeGregori
authors.forename: H Earl
authors.initials: HE
authors.lastname: Ruley
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009355:Neomycin; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000821:virology; D026723:Polyadenylation / Q000235:genetics; D011533:Proviruses / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D014407:Tumor Cells, Cultured; D016662:Virus Integration / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D022821:RNA Splice Sites; C439768:hnRNP A2; D009355:Neomycin