Human embryology week 3

Human embryology week 3

1. Embryonic stem cells

   • Inner cell mass= Embryonic stem cells
   • ES cells come from a blastocyst
   • Oct4 + Nanog + Sox2 expressed: cells keep proliferating as undifferentiated stem cells
   • ES cells in research
     • Helpful to observe when and where a gene is expressed
       • Remove the gene function: assess whether development is affected as expected Hypothesis-driven research
         • Just because a gene is expressed doesn’t mean that its involved in that developmental role.
     • Modified ES cells incorporated into ICM
       • Modified ES cells incorporated into ICM
         • Selection process means most or all are genetically modified

2. Gene targeting

   • First thing is to make your targeting construct.
   • Insert has to have a fragment of DNA of the gene you want to disrupt
   • Insert construct that targets the sequence u are targeting
   • Transfect your plasmid with ES cells
   • Modified gene has inserted copy of the gene with the disruption in place of the endogenous gene
   • Neomycin
     • Neomycin allows first step of the selection process
     • Add neomycin to endogenous cells to kill off any cells that do not have the mutation in the culture
     • Insertion of neomycin resistance in targeting Allows the cells to survive against neomycin
   • Random Integration
     • Gene targeting in mammalian cells
     • Plasmid DNA could insert somewhere else into the genome.
     • These cells will survive neomycin as they have the neomycin resistance but not in the right place
     • Thymidine kinase is inserted when random integration event.
     • Used to do a second round of selection, to eliminate any cells that have the neomycin resistance gene in non targeted areas.
     • Correctly targeted cells when ganciclovir is added are fine and survive. Non targeted cells are all killed at the end of two step process.

3. Conditional knockpouts

   • Target gene is modified: flanked by loxP sites (‘floxed’)
     • Gene is only disrupted in presence of Cre enzyme
       • Gene is only inactivated in cells expressing Cre
         • If you expose DNA to cre enzyme itv causes a recombinations that knocks the gene out.
           • Make a targeted gene but have region flanked with 34bp LoxP sites (“floxed”)
             • Cre recombinase enzyme catalyses recombination between LoxP sites
               • Tandem LoxP sites  deletion
   • First generation: Will have a deletion of one copy of the gene the other copy will be normal Heterozygous conditional knockout
     • Second generation: Half off poffspring will be homozygous for the knockout genes
   • Can also be made:
     • Instead of having cre being driven by a promotor it can be driven by a depended timing control : so cre will only be expressed when the drug is given

4. Transgenic mice

   • Transgenic mice contain exogenous DNA sequence
   • Additional sequence is called a transgene (but can contain several genes) Used to:
     • Express a wild-type gene to rescue a mutant
       • Over-express a gene product
         • Express modified gene products; eg. GFP-tagged proteins
           • Express reporter linked to promoter sequence
             • Express Cre gene linked to promoter sequence

5. Cells that express nanog become epiblast cells : Express FGFR4

   • Cells that express Gata6 become primitive endoderm: Express receptor FGFR2 that recepts FGFR4
   • In the absence of Nanog:
     • ICM cells cannot mature to Epi fate.
       • Without Nanog, cells do not secrete FGF4 to instruct other cells to become PrE.
         • ICM cells form intermediate PrE progenitors but these progenitors cannot mature.

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