Cloning and characterisation of DNA flanking highly expressed integrated plasmids in two mouse myeloma cell lines

摘要

Attempts to optimise plasmid vectors for the high level expression of heterologous proteins in the mouse myeloma cell line, J558L, led to the chance discovery of two highly expressing transfectants (C6 and D8). Both these transfectants resulted from plasmids containing a gpt selection marker, a lysozyme reporter gene and the IgH enhancer. Both expressed the lysozyme reporter at around 100 fold higher level than the normally obtained from transfection with the same plasmids, and approximately the same level as the endogenous immunoglobulin genes.;This thesis describes the cloning and characterisation of DNA flanking the plasmids in both the C6 and D8 cell lines.;A genomic library for the C6 cell line was constructed in -DASH and screened with plasmid sequence probes. A single clone of 15kb was isolated. This contained 9kb of flanking DNA from one side of the plasmid. Sequencing of the clone revealed that the plasmid had integrated within a B1 repeat element. No homologies to other known sequences were identified.;Similarly, a genomic library from the D8 cell line was constructed and screened. Three independent clones were obtained giving a total of 20kb flanking DNA. Sequencing revealed that flanking DNA on one side of the plasmid was extremely A/T rich. The possible implications of this are discussed.;The A/T rich fragment from the D8 locus was shown to bind to isolated nuclear matrix in vitro. However no binding to the nuclear matrix was detected for either locus in an in vivo assay. Both the C6 and D8 loci were shown to be DNase sensitive, but no specific hypersensitive sites were identified for either cell line.;Also presented is work which contributes to a project aimed towards using Flp recombinase to place expression constructs and other DNA sequences at the C6 locus by homologous recombination.