Gene targeting replacement vector

Because of their efficiency in integrating into human DNA, viruses are the most common vectors used to deliver therapeutic genes to recipient cell targets. Disease-causing genes are replaced with the desired therapeutic genes the viral genes that control delivery mechanisms are retained. 

Thomas, K. R., Deng, C., and Capecchi, M. R. (1992) High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol. Cell. Biol. 12,2919-2923. 

Homologous recombination occurs approximately 1000-fold less frequently than non-homologous recombination, therefore methods have been developed to enrich for homologous recombination events. A widely applied method includes the use of a positive and a negative selection marker and does not require the expression of the target gene in ES cells (Mansour et al., 1988). The targeting construct is based on a replacement-type vector... 

Fig. 2. Mutagenesis strategy. A neo gene replaced one-third of the ORF and was used as a positive selectable marker. The HSV thymidine gene (HSV-ffc) was used for negative selection. The vector was linearized with Xbal digestion. The targeted events were screened by Southern blot. Two Xbal sites are in the outside of the targeting vector (the 3 -end one is just at the end of the vector, which cannot be digested by Xbal if the vector is randomly inserted). An outside probe is located at the 5 -end. The genotype of the mice can be easily identified based on the size of the hybridized bands, 6.7 kb (mutant) vs 6 kb (wild-type).

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