CSB349H1 Lecture Notes - Lecture 15: Somatic Cell Nuclear Transfer, Bisulfite Sequencing, Transposase
14 May 2018
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Lecture 15(b): Somatic Cell Nuclear Transfer
Induced Pluripotent Stem Cells (iPSC):
• Direct reprogramming of a differential cell into an iPSC with properties that are very similar
to ESCs only require 4 gene (factors): cMyc, Klf4, Sox2 and Oct4
o Removal of cMyc still allowed for the generation of iPSCs – important because Myc
is an oncogene and can cause tumors if abnormally expressed; however, after the
removal the efficacy of inducing iPSCs reduced dramatically
Experiment:
• Retroviral Transfection System was used to introduce the 4 required
gene into the fibroblast
o Construct: Stem-cell specific promotor (Fbx15) fused with an
antibiotic gene – was used to identify cells that incorporated the
4 genes into their genome
• No Incorporation:
o Gene construct was only transformed into the fibroblast cell and treated with the
antibiotic; cells died because they did not express the antibiotic resistant gene
§ Were incapable of expressing the gene because they are differentiated cells and
cannot activate the Fbx15 promotor
• Yes Incorporation:
o Gene construct and retroviral transfection system transformed into fibroblast cell and
treated with antibiotic; cells survived because they expressed antibiotic resistant gene
§ If a fibroblast cell is turning into an embryonic stem cell fate, Fbx15 promotor
will be active and antibiotic resistant gene is expressed
ð Microarray analysis (gene expression pattern) & bisulfite sequencing (histone methylation) was
completed on iPSCs and ESCs – found to be highly similar but also different (not identical)
o Methylation marks are low and acetylation is high in stem cells
ð iPSCs were used for tetraploid complementation and it was shown that they were capable of
generating a viable embryo; potency of IPSCs is equivalent to ES cells
Inducible & Excisable iPSCs:
• Generate four separate transposon vectors (1 for each factor)
o Transposon vector is transformed with a vector that codes for transposases
§ Expression of transposases can be induced with tetO (doxycycline) treatment
• Transposase will be expressed and induce the transposon vectors to ‘jump’ into the genome
and will begin to express
o Endogenous copies (in heterochromatic regions) will also begin to express; transgene
changes the environment into a stem-cell environment
§ Endogenous copies continue to express and maintain stem cell state even after
the removal of tetO
Document Summary
Experiment: retroviral transfection system was used to introduce the 4 required gene into the fibroblast, construct: stem-cell specific promotor (fbx15) fused with an antibiotic gene was used to identify cells that incorporated the. 4 genes into their genome: no incorporation, gene construct was only transformed into the fibroblast cell and treated with the antibiotic; cells died because they did not express the antibiotic resistant gene. If a fibroblast cell is turning into an embryonic stem cell fate, fbx15 promotor will be active and antibiotic resistant gene is expressed. Ipscs were used for tetraploid complementation and it was shown that they were capable of generating a viable embryo; potency of ipscs is equivalent to es cells. Inducible & excisable ipscs: generate four separate transposon vectors (1 for each factor, transposon vector is transformed with a vector that codes for transposases. Endogenous copies continue to express and maintain stem cell state even after the removal of teto.
