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2-Differential Gene Expression.pdf

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Western University
Biology 3338A
Sashko Damjanovski

•  Genes aren’t destroyed but turned off •  Can regulate transcription at many levels •  Can regulate RNA processing (splice, adding tails etc.) •  Can selectively translate RNA 1 •  Use elements to study how things work •  Look where gene is expressed; if found in certain places, must be transcription factors that are specific to the area (ie. limb, brain) •  If think something is responsible for head, can clone enhancer sequences and hook them up to reporters 2 •  Have pancreatic precursor cellsà can make all parts of the pancreas •  Pdx1 is a pluripotent cell •  Know transcription factors that are needed to make B cell •  If pdx1 is fated to be a B cell, but if give if different transcription factors can end up being something else •  As cells are differentiating they are turning on different TF 3 •  Put a gene into a mouse that isn’t usually there; transgenic mouse usually are a KO of a gene (null) •  Easier to analyze what happens when u KO a gene, than making more of gene •  ICM cells are basically embryonic stem cells •  ICM can form an embryo but not extra embryonic tissue •  ICM cells are pluripotent to embryonic properà can form an entire embryo but not extra tissue •  ICM are not specified •  ICM can be cultured and can add thing to them; can then return them to the embryo without an cells noticing 4 •  Can place vector into cells; want to study bmp, place vector into cells, and grown mice with a lot of bmp •  Usually want to knock out •  Not all embryonic stem cells are going to take in the vectorà vector has a neo resistant gene; use neomycin to kill cells that didn’t take in vector •  Gene needs a promoter; when knocking it out its not an issue 5   •  Put neo r gene into coding of bmpà gene now is not going to code for bmp but for neo r (occurs in plasmid) •  Infect stem cell with plasmid •  Using a variety of enzymes induce homologous recombination with one of the copies of bmp gene •  Will now have one copy of neo r and one of bmp 6   •  Grow cells now in neo •  If don’t have trans gene will die •  Now eject them back into embryo •  Will form tissues that have transgene in them 7   •  Mice that are born contain transgene somewhere in them (random; don’t know where exactly they went) •  End up with chimeric mice (don’t all look the same) •  Some of the tissue in the mice contain transgene (called chimeric because not all tissues are the same) •  Want to knock out gene in every cell (right now they're heterozygous, need homozygous) •  Ideally want transgene to be in gametes so it can be passed onà know if its in gametes by isolating them (this destroys the gametes) •  Do southern blot with bits of the mice, isolate DNA and looking for confirmation that mouse is chimeric •  Neo gene is pre-set that tells us that transgene is present (mice don’t have neo gene) •  Want a homozygous mouse; take chimeric mouse (don’t know if its gametes, but hope) •  If in gametes, 2 possible genotypes (wt or KO) •  Know if its heterozygous (KO) run a blot •  If chimeric mate with other heterozygous sibling 8   •  Equivalent DNA but halve different transcription factors or regulatory factors 9 •  Three different ways of specificationà becomes autonomous •  If a cell is committed and determinedà know what is going to be an
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