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

BackgroundTagged 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.ResultsSequences 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.ConclusionsExpression 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.

Targeted exon skipping of a CEP290 mutation rescues Joubert syndrome phenotypes in vitro and in a murine model

“…RT-PCR analysis with a panel of primers located across the SA-IRES-βGEO cassette suggests that we do not see complete restoration of wild-type transcript levels ( SI Appendix , Fig. S7), possibly due to the presence of cryptic splice sites within the SA-IRES-βGEO cassette (24, 25). However, given that CEP290 is not haploinsufficient and it has been reported that small fragments of CEP290 can ameliorate LCA (26), we reasoned that even subwild-type levels of protein may be beneficial.…”

DJ-1 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress

“…However, the induced activity observed in PON2 deficient MEFs was dramatically lower (less than 5%). The small amount of background lactonase activity observed in PON2 deficient cells may be the contribution of other PON members [60] , [61] , [62] , [63] , [64] , [65] , or the fact that PON2-def mice are reported to having up to 5% of leakiness based on the mouse construction method [66] , although this is unclear at the moment. These results indicate that the lactonase activity induced by DJ-1 is almost exclusively through PON2.…”

Neuronal apoptosis mediated by inhibition of intracellular cholesterol transport: Microarray and proteomics analyses in cultured murine cortical neurons

“…Other proteins of interest include the heterogeneous nuclear ribonucleoproteins, which are involved in pre‐mRNA processing where they form complexes with heterogeneous nuclear RNA (Roshon et al . , 2003) and zinc finger proteins, which belong to the nuclear zinc finger protein family and function as transcription factors (Nagase et al . , 2000).…”