Zhuma, Talgat M.; (1999) Cis and trans elements that influence hCD2 gene expression in transgenic mice. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Locus Control Region (LCR) of the hCD2 gene is able to confer T-cell specific, copy-number dependent and position-independent expression of linked reporter genes in transgenic mice. These properties of the LCR have been exploited for the generation of an expression vector potentially useful in the T-cell specific gene therapy protocols. The hCD2 LCR consists of a strong T-cell specific enhancer and an additional regulatory element without enhancer activity (designated HSS3) which is required for prevention of Position Effect Variegation (PEV) in transgenic mice. To identify trans-acting proteins binding to the HSS3 and, potentially, mediating the LCR function, in vitro footprint analysis of the HSS3 region was performed using thymus and liver nuclear protein extracts. Three of the identified footprinted regions (FT1-FT3) were used as baits in the one- hybrid yeast technique to isolate cDNAs from a T-cell specific library. Using the FT1 sequences as a bait, a 1.3 kb DNA insert coding for the HMG box of the human homologue of rat HBP1 protein was isolated. The rest of the open reading frame (ORF) was isolated from a Lamda-ZAP library. DNA- binding properties of the recombinant hHBP1 was characterised using DNAse I footprint, gel retardation and methylation interference experiments. It was found that HBP1 binds not to a canonical HMG consensus sequence, but to a novel TTCATTCATTCA motif which is higher in affinity than other recently reported HBP1 binding sites. To assess the contribution of FT1 to LCR function, the footprint sequences were deleted from the LCR and transgenic mice carrying a ?FT1-LCR hCD2 minigene were generated. Expression patterns of the ?FT1-LCR hCD2 transgenes were analysed by FACS and the integration sites were identified by FISH and Southern blot. The deletion of FT1 sequences resulted in variegation of expression of the ?FT1-LCR hCD2 transgene when the transgene integrates into heterochromatic regions adjacent to the centromere or telomere. However, the degree of variegation was not as dramatic as one caused by the deletion of the whole of HSS3, indicating that the other HSS3-binding factors may also contribute to prevention of PEV. Sequences of cis regulatory elements other than the LCR were examined for affects on hCD2 transgene expression. This part of the work resulted in an improved version (with 10 x higher expression level) of the hCD2 expression cassette. Subsequently, related experiments have shown that introducing the fourth intron of the hCD2 gene into the improved vector coould result in further 4-5x increase in expression level of the cassette.

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