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Chimeraplasty : gene alterations using DNA/RNA chimeras
In plants and animals, traits can be modified through either induced mutations, and/or recombination or through transfer of foreign genes leading to the production of transgenic plants or transgenic animals. Similarly, in gene therapy, the defective gene is left in its place and the cell is equipped with a new healthy6 gene. In transgenic plants and animals and also in gene therapy, the gene may integrate randomly and not at a homologous site, leading to problems. The presence of a foreign gene in transgenic plants and animals has also come under fire, because the foreign gene remains as a permanent part of the organisms, where it is inserted.
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Chimeraplasty allows alterations in endogenous genes, while maintaining the genomic organization that is important for proper expression/regulation of genes. The alteration of gene in this technology is achieved without the permanent introduction of any foreign DNA. The technique relies on chimeric or hybrid DNA/RNA molecules, that match the target gene region, except for the one base that needs to be altered. By binding to the target gene, a chimera apparently triggers the plant cell’s own DNA repair machinery to correct the mismatch between the target gene and the RNA-DNA hybrid molecule. This leads to the introduction of a base alteration into the gene. The chimeras break down after altering the gene, so that the technique does not leave any foreign DNA within the altered cells.
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Chimeraplasty has its roots in the study of homologous recombination by Eric Kmiec of Thomas Jefferson University in Philadelphia. This process of homologous recombination is efficient only at meiosis, and it has been shown that the rate of recombination rises in active genes being copied into messenger RNAs. This suggested that synthetic RNA may be used in gene repair, so that wild-type DNA may recombine and replace mutant DNA, if added in mutant organism. However, RNA degrades in the cell faster than DNA, so the Kmiec thought of making a hybrid of RNA and DNA and use it for gene repair or also for gene alteration.
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Chimeraplasty : Gene Alterations using DNA/RNA Chimeras
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The technique Chimeraplasty was initially used for alterations in (i) human gene ras to convert it into an oncogene, and in (ii) human gene for liver enzyme alkaline phosphatase. The chimeric molecule used for ras consisted of a five base DNA segment flanked by two 10-base RNA segments. The molecule was a mirror image of ras gene except for a thymine to guanine alteration. Subsequently, in a study published in the year 2000 in mouse, an RNA-DNA oligonucleotide was successfully utilized (in intradermal injection into the skin) to correct a point mutation (TGT to TCT) in tyrosine gene, restoring tyrosinase enzyme activity and melanin synthesis.
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The Tyr-A oligonucleotide was designed to introduce a single base correction (TCT to TGT) in the mutant tyrosinase gene and to convert albino melanocytes to black pigmented cells. This demonstrated a possibility of the use of RNA-DNA hybrid oligonucleotides for skin therapeutics to treat human hereditary skin diseases.
In the year 1999, there were atleast two reports of the use of Chimeraplasty in plants also for altering two endogenous genes, one was herbicide resistance gene and the other was the non-functional green fluorescent protein (GFP) gene. The frequency of alterations in herbicide resistant tobacco plants, treated with chimeric oligos (CO) was 20 times higher than in those not treated; in maize the frequency of these altered plants was two to three orders of magnitude greater than in control plants.
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