Morgenstern, Jay Paul; (1990) Design and construction of mammalian retroviral vectors suitable for cDNA expression libraries. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In an effort to expand the utility of gene transfer/complemetation in mammalian cell culture as a tool in molecular biology, a retroviral based cDNA expression library system has been assembled. An investigation to define the parameters that would maximise clonal gene transfer and reliability of gene expression in retroviral vectors was carried out in order to overcome the deficiencies characteristic of previous vectors which had limited their applicability as cloning vehicles for cDNA expression libraries. The role of sequences within the Mo MuLV genome as cis acting signals for retroviral replication were explored using Mo MuLV based retroviral vectors. This study revealed that sequences both 5' (the splice donor region) and 3' (gag coding sequences) of the previously defined packaging region (ψ site) could potentiate titre of the vectors. Conversely complete deletion of env coding sequences failed to alter vector titre. Based on the above findings, retroviral vectors with an expanded packaging region and multiple drug resistance markers were designed which transmitted inserted genes at titres equal to wild type virus and efficiently expressed them. In order to minimise the possibility of recombination to yield wild type virus during the propagation of recombinant vector helper free producer cell clones, env coding sequences were also excised from these retroviral vectors. A high titre helper free producer cell line of ecotropic host range was designed and built for use in conjunction with these vectors. As model of a mammalian cell culture genetic complementation experiment, an attempt was made to isolate cellular oncogenes that could cooperate with ras in full morphological transformation of primary cells by cotransfecting an F9 teratocarcinoma cDNA expression library, constructed in one of the retroviral plasmid vectors, with an activated c-Ha ras gene into primary rat Schwann cells. In addition several conventional expression vectors were built in which genes could be readily placed under the transcriptional control of different promoters of viral or cellular origin. Transient and stable expression characteristics of the vectors in mammalian cell lines were investigated using a bacterial reporter gene. These vectors were then applied in studies of transformation by Human Papillomavirus 16 and the nature of antigen presentation by HLA class II molecules associated with susceptibility to Tuberculoid Leprosy.

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