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Targetted Gene Transfer-

Targetted gene transfer or gene tar getting uses homologous recombination to replace the endogenous' gene with the functional introduced gene. The first case of such gene transfer (in 1985) was used to disrupt the human l3-globin gene in cultured cells. Subsequently, over 100 mammalian genes have been modified by this approach.

Gene targetting can be used either to inactivate (by disruption) a functional endogenous gene or to correct a defective one. The vectors employed for gene targetting are of two types:

(i) insertion vectors and
(ii) replacement vectors.

The insertion vector is linearized by restriction cleavage within the sequence to be targetted; the targetted sequence provides the site for recombination and is different from the gene to be introduced. Hence the sequence to be introduced is located in the inner region of the vector and is flanked by the sequences involved in recombination.

A recombination of such a vector with its homologous cellular sequences produces a duplication of the targetted sequence; this is called Insertional recombination. In contrast, a linearized replacement vector has the two halves of the target gene at its twp ends.

Recombination occurs within the two halves of the target gene, replacing a portion of the endogenous gene sequence by that of the introduced gene: this is called replacement recombination. There is no duplication of sequences, and the target gene becomes disrupted.

A strategy has been devised to modify only a small sequence of the target gene without the attendant gene duplication/disruption produced by insertional/replacement recombination. This approach, called in out method of gene tar getting, consists of the following two steps.

1. The first step called "in" step, is targetted gene transfer using an insertion vector; the appropriately targetted cell will have a gene duplication.

2. The second step, termed as "out" step, depends on either intrachromosomal recombination (between the introduced and the endogenous genes) or unequal sister chromatid exchange between homologous chromosomes. The recombination product of interest is a chromosome, which has only a single and functional copy of the introduced gene.

Gene targetting is the strategy of choice for gene therapy for the following reasons.

(i) The targetted gene is changed in a precise and specific manner.
(ii) The introduced functional gene is placed in the same context, i.e., is flanked by the same DNA sequences, as the replaced endogenous gene. And

(iii) no other gene of the genome is affected. The major limitation of the approach is the low frequency of homologous recombination, but it is expected that targetted therapy would become a feasibility for many genetic diseases in the near future.

 
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