CSB349L18 - CSB349 Lecture 18 Reprogramming Somatic cell...

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CSB349 Lecture 18 November 22 nd Reprogramming: Somatic cell nuclear transfer Map where SNPs are and look at same gene and see if the SNPs are from one nucleus vs. the other. Design your own transgenes that express a reporter that will only be expressed in certain cells, like neurons. If the cell is being reprogrammed, the gene will actually be expressed, otherwise not, For reprogramming to occur, the cell must be producing a microenvironment that induces changes. The donor cell will adapt to the new environment. In circumstances where you can get nuclear reprogramming without cell fusion, you need the right environment (phenotypic plasticity). You have to rule out cell fusion first. Another way of doing this is to do technique of nuclear transfer. Just take out nucleus and move it to another cell. The nucleus can be reprogrammed. The donor nucleus, if you take it from an advanced stage of differentiation, it is less manageable to be reprogrammed, unlike an embryonic cell. Differentiated doesn’t work too well. The frequency with which this experiment is successful (taking a nucleus from an existing cell, putting it in another organism), is very very very low. 1%. Maybe the only time you get success is when you transfer over a stem cell nucleus without you even knowing (progenitor cell).
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CSB349 Lecture 18 November 22 nd The amphibian egg is large relative to most mammalian eggs. Difficult in mice. In early experiments, they used mouse eggs but kept the nucleus of the egg and transferred over a new nucleus. Not so great success with this. Then they copied the standard amphibian approach which was to first enucleate the host then add the new nucleus. One approach was to use the zygote instead of the egg as a host (fertilized egg instead), the timing is so short as the embryo environment should be as permissive as the zygote environment. Zygote host was not as good though. Now this technique does work. Number of technical modifications to make it work (clone sheep). The egg cytoplasm is the most permissive microenvironment for a new diploid nucleus to be inserted. The early zygote CAN be a reasonable host but it has to be the right environment (e.g. when arrested at metaphase). But egg environment is best. You do need to remove the nucleus first b/c ploidy is a big issue. The embryo proper has to be diploid.
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This note was uploaded on 12/03/2010 for the course CSB CSB349 taught by Professor V.tropepe,a.moses during the Fall '10 term at University of Toronto.

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CSB349L18 - CSB349 Lecture 18 Reprogramming Somatic cell...

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