CSB349H1 Lecture Notes - Lecture 15: Somatic Cell Nuclear Transfer, Cre Recombinase, Open Reading Frame
14 May 2018
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Lecture 15(a): Somatic Cell Nuclear Transfer
Question 1 – Is reprogramming of somatic cells possible?
• Must ensure that the nuclear donor cells are fully differentiated – was done by:
o GFP marker was fused with the promotor; Olfactory Marker Protein (OMP) –
therefore, only fully differentiated cells will exclusively express the protein to drive
GFP expression
• Created two transgenic lines of mice which were then crossed with each other: =
a) OMP promotor driving the expression of Cre Recombinase
b) b-Actin promotor driving expression of B-geo (antibiotic gene) which is flanked with
P-sites followed by a GFP open reading frame
ð Cre-Lox System:
o Cre Recombinase will infuse with the Lox-P sites inducing them to ‘jump’
(similar to transposons)
§ When Cre Recombinase and Lox-P sites come together, Lox-P will jump out
of the DNA – generate a new construct where antibiotic gene is no longer
present and GFP can be expressed
• After crossing both transgenic lines together, all cells will have both constructs:
o Non-Differentiated Cells:
§ Will not express Cre Recombinase – therefore,
will not induce Lox-P jump, no GFP expression
o Differentiated Cells:
§ Will express Cre Recombinase – therefore, Lox-P
jump occurs and will see GFP expression
Experiment:
A) Getting a Tetraploid Cell & ESCs:
o Nucleus of fully differentiated cell was injected into an enucleated oocyte
§ Oocyte will begin to undergo cellular division and
prior
to blastocyst
formation the cells are treated with an electric pulse causing
two cells
to fuse
generating a tetraploid cell
• Once the oocyte reaches the blastocyst stage, ESCs are isolated from
the inner cell mass
B) Tetraploid Complementation ~ testing pluripotency characteristics:
o Isolated ESCs are injected into the tetraploid blastocyst – if capable of obtaining viable
embryos then it must have been derived from the ESCs
§ Tetraploid cells cannot develop further; embryo can only develop from
diploid cells
ð Addresses the question that viable
offspring are capable of being
produced from fully differentiated cells
Document Summary
Gfp expression: created two transgenic lines of mice which were then crossed with each other: , omp promotor driving the expression of cre recombinase, b-actin promotor driving expression of b-geo (antibiotic gene) which is flanked with. P-sites followed by a gfp open reading frame. Cre-lox system: cre recombinase will infuse with the lox-p sites inducing them to jump" (similar to transposons) Will not express cre recombinase therefore, will not induce lox-p jump, no gfp expression: differentiated cells: Will express cre recombinase therefore, lox-p jump occurs and will see gfp expression. Experiment: getting a tetraploid cell & escs, nucleus of fully differentiated cell was injected into an enucleated oocyte. Tetraploid cells cannot develop further; embryo can only develop from diploid cells. Addresses the question that viable offspring are capable of being produced from fully differentiated cells. Suggests that epigenetic inheritance may be the cause of this problem.
