Retroviral Insertion

Retroviruses can be modified by recombinant DNA techniques to make them replication-defective and to replace part of the viral DNA with a desired transgene so it can then be used as a gene vector. Retroviral-mediated gene transfer was originally used to insert transgenes into mice embryos[5] and blostodermal cells of chicken eggs.[6] In comparison to microinjection, retroviral infection offers the following advantages: 1) integration of single copies of the gene; and 2) retroviral DNA integrates into a high percentage of embryos when exposed to high concentrations of viral stock by coculture with infected cells in vitro, or in the case of chickens, by microinjection into the blastodisk. The disadvantages are: 1) it requires added work to produce a retrovirus carrying the transgene; 2) the gene being transferred must be smaller than 10 kb in size; 3) resulting transgenic animals are frequently highly mosaic, which necessitates extensive outbreeding to establish pure transgenic lines; and 4) unresolved problems with hypermethylation can interfere with expression of the transgene.

More recently, retrovirus-mediated gene transfer has been used to produce transgenic cattle by insertion of retroviruses into metaphase II oocytes to avoid mosaicism and ensure that a high percentage of the offspring are

transgenic.

Many laboratories involved in the production of transgenic livestock have not embraced the use of retroviral insertion technology because of concerns about public perception and the potential consequences of recombination events between the viral vectors and endogenous retroviruses to generate new pathogenic agents.

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