Immortalization of human chorionic villi cells with plasmid pSV3 Neo containing simian virus 40 T antigen

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Immortalization of human chorionic villi cells with plasmid pSV3 Neo containing simian virus 40 T antigen

 

Author: Pang, Chung-wah Kenny
Title: Immortalization of human chorionic villi cells with plasmid pSV3 Neo containing simian virus 40 T antigen
Year: 1999
Subject: Human cell culture
Cell transformation
Plasmids -- Genetics
Genetic engineering
Biotechnology -- Research
Hong Kong Polytechnic University -- Dissertations
Department: Multi-disciplinary Studies
Dept. of Applied Biology and Chemical Technology
Pages: ix, 65 leaves : ill. (some col.) ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1460554
URI: http://theses.lib.polyu.edu.hk/handle/200/551
Abstract: In this experiment, it was attempted to transform human chorionic villus (CV) cells donated by Tsan Yuk Hospital into an immortalized cell line by the plasmid pSV3 Neo containing the simian virus T antigen. The CV cells were cultured in AminoMax-C100 medium, which was developed specifically for the in vitro prenatal diagnostic testing of human amniotic fluid specimens. They were found to be able to propagate for about 40 passages. The E. coli harbouring plasmid pSV3 Neo obtained from American Type Culture Collection was reconstituted with LB medium and the identity of the plasmid extracted was confirmed by restriction digestion followed by agarose gel electrophoresis. Plasmid DNA extracted by manual method was then used for transfection process with Lipofectin R reagent; however, no transformed cells could be obtained. Extractions by two commercial kits were made to yield plasmid DNA of higher purity. Second transfection using plasmid DNA extracted by commercial method produced transformed cells but they died shortly in selection medium made from AminoMax-C100 plus G418. Two other culture media, namely, F-12 and DMEM, together with AminoMax-C100 were used to optimize the growth of the transformed cells produced in the third transfection. All three media were prepared with a different percentage of serum/supplement to test the effect of medium composition as well as serum concentration on the growth of the transformed cells. The transformed CV cells were found best supported by DMEM with 10% FCS. The 'normal' CV cells showed anchorage-dependence and were long and elongated in shape. Contrarily, the transformed CV cells lost their anchorage-dependence and formed a suspension culture in the selection medium. They were small, round in shape and quite transparent in the beginning. Later, they gained bigger in size and darker in colour which were probably due to increased DNA synthesis. After 6 weeks of continuous cultivation, they started to grow slowly and were awaiting further characterization.

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