A gene knockout for the telomere protein TRF1, in the chicken cell line DT40

• Main Author: Baird, K. M.
• Published: University of Cambridge 2000
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596269

Telomeres exist at the ends of eukaryotic chromosomes, as a complex between simple DNA repeats and associated proteins. Members of the 'telobox' family of proteins share a functional domain for binding double-stranded telomeric DNA. One such protein from human, TTAGGG repeat binding factor 1 (TRF1), has been implicated in the regulation of telomere length. This thesis details the characterisation of the chicken homologue of TRFI, the subsequent knocking-out of gene function in the chicken B-cell derived line, DT40, and analysis of resulting cell lines. cDNA and genomic clones of chicken TRF1 have been characterised. The predicted protein sequence is highly related to human and mouse, especially in the conserved functional domains for telomere binding and for protein homodimerisation. Polyclonal antibodies raised to the chicken TRF1 sequence localise to chicken telomeres, as does ectopically expressed FLAG-TRF1 fusion protein. The gene has been mapped by FISH to chicken chromosome 2. The genomic organisation of chicken TRF1 has been determined and this information used to build knockout constructs. Levels of homologous recombination are much higher in DT40 than in any available human or mouse somatic cell lines, so this line was used for targeted gene replacement of the TRF1 gene. DT40 lines deleted for 1, 2 and 3 (all) copies of TRF1 have been produced. These have been assayed for telomere length, karyotype stability, <I>de novo</I> telomere-seeding competence and growth rate. Despite complete inactivation of TRF1 protein, none of the parameters measured appeared to differ from those in wild-type DT40. The implications of these results are discussed.