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LCR-activated expression of cloned K+ channel genes in mammalian cells.

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thesis
posted on 19.11.2015, 09:07 by Philip Andrew. Shelton
The human beta-globin LCR confers high levels of expression on cis linked cDNAs and genes under control of the beta-globin promoter, and certain genes under control of their native promoter elements. This heterologous expression is copy-number-dependent and position-independent in the nucleii of erythroid specific cells. Mouse erythroleukemia (MEL) cells have been established as being electrophysiologically quiet with respect to K+ channels and suitable for patch-clamp analysis. Furthermore, the MEL-cell line has been utilized as an erythroid-specific host for the stable expression of mammalian delayed rectifier (RCKl), transient (A-type) {lcub}RatShal and hPCN2) and inward rectifier type (IRK1) voltage-activated K+ channel cDNAs under control of the beta-globin promoter. Parallel work has allowed the characterisation of a novel human delayed- rectifier type K+ channel (hCDRl) from human chromosomal DNA, and an analysis of the hCDRl gene promoter region has been initiated. Furthermore, a 5.2kb Bg/II-generated DNA fragment (hCDR15.2) containing the full hCDRl coding-region and 2.4kb of upstream chromosomal sequence was isolated. hCDR15.2 has been linked to the human beta-globin LCR and activation of native hCDRl promoter elements in MEL-cell nucleii and subsequent detection of expressed hCDRl homotetramers was achieved by patch-clamp analysis. Finally, the establishment of a gene-activation library for the functional expression of novel and existing K+ channel genes has been attempted. A rabbit genomic DNA cosmid library was constructed with firstly a 1kb (nanolocus) LCR cassette shown to confer 30-40% full LCR activity and secondly a full LCR (6.5kb) based vector. 14 different nanolocus-based rabbit genomic cosmid library clones, shown to be homologues of voltage-activated K+ channel sequences, were isolated. No detectable expression was observed from any of these clones after stable integration into MEL-cell nucleii. Transcriptional silencing and insulation have been discussed as probable reasons for the lack of detectable expression from these 14 clones.

History

Date of award

01/01/1993

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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