embryonic stem cells.docx

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Biological Sciences
Ken Cho

-embryonic stem cells : pluripotent; grown in petri dish; inject into blastocoels mouse cavity IcM cells of blastocyst remain pluripotent inject back into host mother offspring will be chimaeric mutants; want some of the mutated cells to go into the germ cells; ultimately want to make a pure mutant heterozygote from the embryonic stem cell line (that shows a deficiency in a given gene); this is how we obtain knockout targeting vector drug resistant gene =toxin gene; inactivate the gene function upon homologous recombination, it will create resistance interesting knockout mice myostatin; inhibits muscle growth; causes overgrowth of muscle development; Clicker question: mate the following chimera male with a wild type albino female (white mouse). What percentage of the offspring will have brown coat color? Answer: impossible to predict -some sperm contain the mutant gene and some sperm might not contain the mutant gene -we cannot predict the outcome when mating a chimeric male with a female conditional knockout -some genes are specifically expressed and others can be ubiquitously expressed (expressed everywhere) -if we knock out the gene function, we will get a dead embryo -wanted to express a knockout gene in a specific tissue at a specific time +use Cre0IoxP system +lox P located on both sides of the gene: in the presence of Cre (a recombinant enzyme), it would cleave the loxP site = consequence would be the target gene would have complete deletion of the final product +experiment: tried to express Cre only in the eyes and mated the Cre mouse with the LoxP mouse ~result: gene is knockout out in the eye but not in the other genes b/c Cre is only expressed in the eyes; this is a conditional knockout used to create a specific knockout in a specific gene how to determine when and where a gene’s mrna is present -how do we detect the amount of transcript in an embryo? +embryonic cells contain different types of transcripts +make antisense RNAthat recognizes the complement and label it with isotope or fluorescent dye +but it will not touch the other transcripts not complement to it; only will form hybrids with the complements, wash away the other transcripts, and look at the hybrids under the fluorescent microscope. Can tell what kind of genes are expressed in which tissues. Lec 9: setting up body axes -Wnt signaling include the growth factor superfamily also -the growth factors influence the expression of the transcription factors to make a gene setting up body axes -has polarity; animal side = bla
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