Tsakiridis, Anestis

2018-03-29T12:20:45Z

2018-03-29T12:20:45Z

2007

Gene trapping is a random insertional mutagenesis strategy that aims to identify novel genes and analyse their function. It usually involves the introduction into embryonic stem (ES) cells of promoterless reporter/selector gene constructs whose expression can be activated only after integration downstream of a gene's regulatory elements. Gene trap insertions result in production of fusion transcripts consisting of the reporter and endogenous sequences and the mutated genes can be readily identified using PCR-based methods such as RACE. Furthermore the biological consequences of the integration event can be assessed after germ-line transmission. One limitation of conventional gene trapping is that it can only target genes expressed in ES cells since selection of insertional events relies on the endogenous promoter's activity to drive expression of the selectable marker and to circumvent this problem a new class of gene trap vectors called poly(A) trap vectors was developed. These constructs contain a 3' selectable marker whose expression is driven by a constitutively active internal promoter relaxing the requirement for endogenous gene expression. The selectable marker lacks a polyA signal but incorporates a splice donor (SD) signal so only integrations upstream of an endogenous gene's splice acceptor (SA) and polyA sequences can be selected thus eliminating intergenic background insertions. However, it has been recently demonstrated that poly(A) trap vectors are biased towards integrations into the 3'most-intron of their target genes due to the action of an mRNA-surveillance mechanism called nonsense-mediated mRNA decay (NMD).

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, P-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 contains the previously uncharacterized rabbit (3-globin exon 2/intron 2 SD junction and an AU-rich element (ARE) derived from the human GM-CSF gene. Our results provide evidence that the triple fusion functions properly and can be potentially used as a reporter of trapped locus activity. We also show that the presence of the ARE appears to improve the performance of the rabbit (3-globin SD sequence in the context of poly(A) trapping. More importantly, preliminary data suggest that our vectors may be resistant to NMD and thus potentially unbiased in their insertional preference.

http://hdl.handle.net/1842/29405

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 17

Already catalogued

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

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy