Transfer of genes to fertilized eggs or embryos. The transfection of fertilized egg may involve transfer of whole nuclei, whole chromosomes (or fragments) or DNA segments, (i) For the
transfer of whole nuclei, the egg cells are treated with
cytochalasin-B and subjected to centrifugation causing enucleation (nucleus is removed). Incubation of desirable karyoplasts with these enucleated eggs leads to the transfer of whole nuclei in presence of polyethylene glycol (PEG); (ii) For
transfer of whole chromosomes, the chromosomes are first isolated from metaphase cells by hypotonic lysis and may be fractionated using density centrifugation or
flow cytophotometry. Individual chromosomes or fragments thus isolated are then incubated with whole cells (eggs) for incorporation of chromosomes into nuclei; (iii)
Microinjection of DNA segments into fertilized eggs is the most commonly used method (Fig. 42.1). Several hundred copies of the DNA segment are utilized and after stable integration, the eggs are used for getting transgenic animals. This method is commonly used for the production of transgenic mice (Fig. 42.2).
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| Fig. 42.1. Microinjection of foreign DNA into a fertilized egg for transfer of a gene leading to the production of transgenic animals. |
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| Fig. 42.2. Different steps in the production of transgenic mice through microinjection of eggs. |
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Transfer of genes to cultured cells.
Transfection of cultured cells (usually stem cells; stem cells are undifferentiated precursor cells) is often attempted for purposes of gene therapy, etc. but can also be used for production of transgenic plants. Gene transfer to cultured cells can be achieved either through the use of retroviruses and other viruses as vectors or by direct delivery of DNA by any of the following methods (Fig. 42.3) : (i) coprecipitation of DNA with calcium phosphate; (ii) use of complexes, of DNA with polycations or lipids; (iii) electroporation (a technique, where cell membrane is made more permeable by electric shocks); (iv) use of gold or tungsten particles coated with DNA and accelerated to a high speed using a particle gun (see later); (v) microinjection of DNA using micro-manipulator and (vi) use of liposomes or sphaeroplasts, which ar vesicles carrying DNA. In future more efficient methods of gene transfer to cultured cells in animals will become available. |
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| Fig. 42.3. Transfection of stem cells and injection of derived stem cells into blastocyst, leading to the production of transgenic |
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