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A mutational analysis of the regulation of retroviral transcription in embryonic stem cells

Singh, Devender; (1994) A mutational analysis of the regulation of retroviral transcription in embryonic stem cells. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Viral genes provide a useful model system for studies of transcriptional control in embryonic stem cells. The Moloney murine leukemia virus (Mo-MuLV) and its close relative Moloney murine sarcoma virus (Mo-MSV) are incapable of productively infecting embryonic stem cells. Since, the viral long terminal repeat (LTR) sequences are unable to direct transcription in embryonic stem cells but are highly active in differentiated derivatives, these viruses provide a useful probe for investigating transcriptional control in embryonic stem cells. Barklis et al. (1986) identified the tRNA primer binding site (PBS) as a negative regulator of provirus expression. Prince and Rigby (1991) observed in transfection experiments that generation of an Sp1 binding site by introducing a point mutation at -166 (bp) with respect to the transcriptional start increased transcription 6-fold. Tsukiyama and Niwa (1992) analyzed an EC-specific repressor activity and identified a consensus sequence for the binding of this repressor protein. In this study, mutations in the negative regulatory element at the PBS, in the binding site for the EC-cell specific repressor and at the mutated Sp1 binding site were analysed in retroviral transduction experiments in embryonal carcinoma and embryonic stem cells. I found that infection with the MSGLacZ vector is an effective permissive delivery system in the absence or presence of any mutation in EC and ES cells. Retroviruses with single, double and triple mutations in different combination of mutated repressor sites and a generated Sp1 site were produced using [psi]Cre producer cells. Maximal expression was obtained when the virus carries two mutations (MSGLacZ, -166, B2) in which an Sp1 binding site is generated and the B2 repressor site is blocked. The MSGLacZ (-166, B2) virus not only increased expression 4-fold in EC and 8-fold in ES cells but also expressed efficiently in the inner cell mass of the blastocyst stage embryo. Two repressor sites were identified in the primer binding site region and the other EC-specific repressor was found not to be effective. A new method of transfection, which increases transient transfection efficiency more than 122-fold in neuroblastoma cells was developed. A 4-fold increase in stable clones was also obtained when BAGLacZ, neo was transfected into ψCre cells. New ionic substrates were developed to replace polybrene due to its toxicity and to increase the transduction efficiency of retroviruses in EC and ES cells.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A mutational analysis of the regulation of retroviral transcription in embryonic stem cells
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Embryonic stem cells; Retroviral transcription
URI: https://discovery.ucl.ac.uk/id/eprint/10102995
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