Targetted Gene transfer or gene targetting uses homologous recombination to repalce the endogenous gene with the functional introduced gene. The The first case of such gene transfer was used to disrupt the human B globin gene in cultured cells over through this approach more than 100 mammalian found through this sequence 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 duplication of the targetted sequences this is called insertional recombination. A strategy has been devised to modify only small sequence of the largest gene without the attendant gene duplication produced by insertional recombination. This approach, called in out method of gene targetting consists of the following two steps.
The first step called "in" steps, is targetted gene transfer using insertion vector the appropriately targetted cell will have gene duplication.
Second step termed as "out" step, depend on either intrachromosomal recombination or unequal sister chromatid exchange between homologous chromosome the recombination product of interest is chromosomes which has only a single and functional copy of the introduced gene.
The in-out strategy has been tested using the HGPRT gene. The gene was targetted into a mouse embryonic stem cell line subsequently it has been succsfully used with some other gene. This procedure is ideal for gene therapy.
Gene targettin is the strategy of choice for gene therapy for the following reasons.
(1) The targetted gene is changed in precise and specific manner.
(2) The introduced functional gene is placed in the same context it is flanked by the same DNA sequences as the replaced endogenous gene.
(3) No other gene of the genome is affected. The major limitation of the approach is the low frequency of homologous recombination this problem however is being removed by refinments of the teqchique. The feasibility of gene targetting has been demonstrated in number of different cell tyupes for several different genes. It is expected that targetted therapy would become a feasibility for many genes disease in the near future.
The first step called "in" steps, is targetted gene transfer using insertion vector the appropriately targetted cell will have gene duplication.
Second step termed as "out" step, depend on either intrachromosomal recombination or unequal sister chromatid exchange between homologous chromosome the recombination product of interest is chromosomes which has only a single and functional copy of the introduced gene.
The in-out strategy has been tested using the HGPRT gene. The gene was targetted into a mouse embryonic stem cell line subsequently it has been succsfully used with some other gene. This procedure is ideal for gene therapy.
Gene targettin is the strategy of choice for gene therapy for the following reasons.
(1) The targetted gene is changed in precise and specific manner.
(2) The introduced functional gene is placed in the same context it is flanked by the same DNA sequences as the replaced endogenous gene.
(3) No other gene of the genome is affected. The major limitation of the approach is the low frequency of homologous recombination this problem however is being removed by refinments of the teqchique. The feasibility of gene targetting has been demonstrated in number of different cell tyupes for several different genes. It is expected that targetted therapy would become a feasibility for many genes disease in the near future.
