Gene transfer in mammalian cells

Gene Transfer in Mammalian Cells
During the 1980's, techniques in mammals were also developed for transfer of genes, which were either isolated from native cells or synthesized organochemically. The genes may either be isolated and inserted in the form of whole chromosomes or chromosome segments, or may be used in the form of DNA samples used for transfection of isolated host cells in culture (for more details, see Genetic Engineering and Biotechnology 3.  Isolation, Sequencing and Synthesis of Genes).

Chromosome mediated gene transfer
A chromosome from a mammalian cell can be isolated by differential centrifugation of carefully broken cells, whose mitotic cycle has been arrested at metaphase by the drug colchicine, since metaphase chromosomes are relatively robust and are easy to be isolated. These isolated chromosomes are allowed to be taken up by cells in culture and success has been achieved in some cases where individual human chromosome 17 or a segment, could be transferred to a mouse cell line. This chromosome 17 thus transferred carried the gene for thymidine kinase and therefore its transfer could be checked on HAT medium, discussed earlier for chromosome mapping. The chromosome segment with markers, was later found to be translocated to a mouse chromosome. This process is parallel to the bacteriophage mediated transduction in bacteria discussed in Sexuality and Recombination in Bacteria and Viruses.

Transformation of cells with free DNA
Genes in mammalian cells can also be transferred from solutions of free DNA i.e. by transformation discussed in Sexuality and Recombination in Bacteria and Viruses and Genetic Engineering and Biotechnology 4.  Gene Transfer Methods and Transgenic Organisms. It has been possible to transform TK^- (thymidine kinase deficient) mouse cell into TK⁺ cells by transformation, where HAT medium was used for screening. The transforming potency of DNA could be increased by digestion of DNA with a restriction endonuclease and then fractionating fragments by size, so that in one case a fragment size of 3.4 kb was more effective in transforming TK^- cells into TK⁺. Even then the frequency of transformation was one in one million, but is useful in cultures, where large populations of cells can be handled. Another example of transformation is the transfer of rabbit β globin gene to mouse cells which were TK⁺ and were, therefore, earlier selected for their transforming ability. In most cases the foreign DNA in these cases gets integrated into mammalian chromosomes. This transformation can rarely be brought about in the fertilized mouse eggs also, and it was shown that rabbit β globin was produced by the mouse developed from transformed egg and that the rabbit β globin gene thus introduced is inherited to the next generation.