Recitation 16 Transgenesis editing a gene in an organism Knockouts KO usually loss of function In a mouse Example If the knockout phenotype is a tumor then we can see that the gene is a tumor suppressor The gene is always the opposite of the loss of function phenotype 1 Obtain a blastocyst 2 Culture embryonic stem cells ES cells in vitro 3 Make a construct to edit the gene of interest 4 Add edited ES cells to a host blastocyst 5 This give rise to a chimera mosaic mouse 6 Hope that the edited cells gave rise to the germ line 7 Mate the heterozygous knockout mice together to make homozygous knockout mice which we can then study Conditional Tissue Speci c Knockout To have control over spatial or temporal expression Done using a cre lox system Cre is an enzyme that will ip out a gene at speci c sites know as lox sites We use promotors to promote the expression of Cre RNA Interference Knockdown causes RNA to be degraded so it cannot encode for a protein Less effective than a knockout Rather than deleting a gene it destroys the RNA 1 Inject double stranded RNA into organisms 2 Use RNA directed RNA polymerase in order to amplify the RNA 3 Binds to all complementary mRNA 4 Cellular mechanisms seek and destroy mRNA hybrids Argonaut pathway This helps counter viruses microMRNAs are used by cells to control gene expression
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