Assessing the efficiency of novel gene trap vectors in murine embryonic stem cells

• Main Author: Tsakiridis, Anestis
• Published: University of Edinburgh 2007
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663067

The aim of the study presented here was to assess the efficiency of a series of gene trap vectors that incorporate two novel features in their design: (i) the presence of an ATG-less, 5’ triple fusion between egfp, beta galactosidase and neomycin/hygromycin resistance genes to function as a reporter/selector of the trapped gene’s expression state and (ii) a 3’ poly(A) trap cassette that consists of the constituttive human β-actin promoter, the neomycin resistance gene and the rabbit β-globin exon 2/intron 2 SD junction. An AU-rich element (ARE) was also included to further promote selection of integrations into transcriptional units. The results of the experiments that were designed to characterise these features demonstrate that: (i) the 5’triple egfp/βgal/neo^r or hygro^r fusion can be successfully employed as a tag for trapped gene activity mainly in the case of transcriptional units expressed at high levels. Surprisingly 5’RACE PCR analysis of the resulting gene trap insertions showed that in many cases reporter protein translation as evidenced by X-gal staining and FACS/microscopy occurred despite the presence of in frame stop codons in the upstream endogenous, trapped sequence. (ii) the efficiency of the vectors’ poly(A) trap was compromised by the presence of a cryptic SA site present within the vector backbone and an improvement in the performance was observed after employing a ‘corrected’ vector version in which the cryptic SA was removed. The presence of an ARE facilitated further enhancement in the vector’s mutagenic potential.