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TARGETED GENE THERAPY
| Targeted gene transfer or gene targeting uses
homologous recombination to replace the endogenous gene with the
functional introduced gene. The first case of such gene transfer
was used to disrupt the human beta globin gene in cultured cells.
Subsequently over 100 mammalian genes have been modified by this
approach. gene targeting can be used either to inactivate a
endogenous gene or to correct a defective one. The initial gene
targeting to disrupt the human beta globin gene used a double
selection strategy called positive negative selection. |
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The vectors employed for gene targeting are of
following two types:
- Insertion vectors,
- Replacement vectors
Insertion vector is linearised by restriction cleavage within the
sequence to be targeted. The sequence is located in the inner region
of the vector & is flanked by the sequences involved in recombination,
this produces a duplication of the targeted sequence. Hence called
insertional recombination. |
A linearised replacement vector has the 2 halves of the target gene
at its 2 ends. recombination occurs within the 2 halves of the target
gene, replacing a portion of the endogenous gene sequence by that of
the introduced gene. Hence called replacement recombination. The
target gene becomes disrupted, hence, this approach can't be used for
gene therapy.
A strategy has been devised called as In-out method of
gene targeting to modify only a smal sequence of the target gene.
It consists of 2 steps.
- The first step is In step, where the targeted gene is
transferred using an insertion vector.
- This is the Out step which depends on either
intra-chromosomal recombination or unequal sister chromatid
exchange between homologous chromosomes.
This strategy has been tested using HGPRT gene. The gene was
targeted into a mouse embryonic stem cell line. |
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