Retroviral vector mediated gene trapping in mice

There is a great wealth of sequence information available nowadays, but this is accompanied by a serious lack of functional information. Functional genomics is an area that needs to be developed and one method being utilised is gene trapping. In this project a gene trapping approach was employed to achieve insertional mutagenesis in vitro in embryonic stem (ES) cells. These early embryo derived cell lines can be manipulated in vitro and then returned to the embryo where they participate in the normal development of a chimeric mouse. There are two types of gene trapping retroviral vectors being investigated, one is a shuttle vector which contains plasmid backbone between the long terminal repeats (LTRs) allowing the rescue of any trapped gene. The other is a splice acceptor (SA) vector, which has a SA in front of a promoterless P-geo gene. The provirus integrates into the genome of the ES cells and the trapped gene is tagged with the p-geo reporter gene. This enables trapped clones to be selected with G418 and expression patterns to be visualised by staining for p-galactosidase activity. Generation of a fused RNA transcript between the trapped gene and vector sequences facilitates cloning of the trapped gene. Using the P-geo sequence of the integrated vector, 3' RACE PCR was used to amplify a segment of the trapped gene and subsequently obtain sequence data. Two retroviral vectors of each type mentioned are examined in this project for their insertional mutagenesis ability in vitro in ES cells and subsequent analysis. Neither of the shuttle retroviral vectors gave any reproducible results. A comparison was made between the SA retroviral vectors, one containing an internal ribosome entry site (IRES), the other not. These vectors gave rise to resistant ES cell clones and subsequent 3' RACE PCR sequence data