The expression of glutamate dehydrogenase in CZB cultured mouse embryos : a biology departmental honors thesis [(HONRS 499)]

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Martin, Emily P.
Chatot, Clare L.
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Thesis (B.?.)
Honors College
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CZB medium, which contains glutamine at a concentration of 1 mM, has been developed to culture mouse embryos from the 1-cell stage to the blastocyst stage (Chatot et al., 1990). Studies indicate that glutamine is essential to the development of some strains of mouse during preimplantation stages in vitro and it is used in vivo as an energy source (Chatot et al., 1989). Glutamate Dehydrogenase (GDH) is an enzyme that is necessary for the metabolism of glutamine. The purpose of our research is to determine the amount of GDH expressed, via the analysis of mRNA, in preimplantation mouse embryos that have developed to various stages both in vivo and in CZB culture. My specific goal is to analyze the GDH mRNA levels of those embryos that have been cultured in CZB medium using the technique of RT-PCR. All culture experiments had greater than 54% developing to the blastocyst stage, with the average percent developing into blastocysts being 78.5%. The overall mean of cells per embryo was 36.6 ± 1.85 (n=195), which is consistent with previous in vitro results by Chatot et al. (1990). Mutant GDH (mGDH) internal control plasmid has been isolated from DH5a bacterial cells and subsequently linearized for RNA transcription via digestion with Hind III. In future experiments, mGDH internal control RNA will be transcribed from the pGDHmJL plasmid. RNA from CZB cultured embryos spiked with mGDH RNA will be subjected to quantitative RT-PCR to determine the developmental expression of endogenous GDH.