Embryonic stem cells and homologous recombination

Question 1

What are embryonic stem cells isolated from?

Answer 1

-Isolated from the inner cell mass cells of a blastocyst

Question 2

What are embryonic stem cells isolated from?

Answer 2

-Isolated from the inner cell mass cells of a blastocyst

Question 3

What type of cells are inner cell mass and what can they form?

Answer 3

-Inner cell mass are pluripotent and can from whole animal when transferred back into an embryo

Question 4

How do embryonic stem cells proliferate in culture?

Answer 4

-Will proliferate indefinitely in culture (and remain pluripotent, under the right conditions)

Question 5

How do embryonic stem cells proliferate in culture?

Answer 5

-Will proliferate indefinitely in culture (and remain pluripotent, under the right conditions)

Question 6

How are chimeras generated?

Answer 6

• Targeted embryonic stem cells are injected into a blastocyst
• Those blastocyst with the embryonic stem cells will be implanted a foster mother
• Those embryonic stem cells will integrate at random into the developing embryo
  
  • You may get none or a lot of contribution of embryonic stem cell
• Cells/tissues derive from either targeted embryonic stem cells or wild type cells

Question 7

What are chimera?

Answer 7

A chimera is essentially a single organism that’s made up of cells from two or more “individuals”—that is, it contains two sets of DNA

Question 8

How does homologous recombination work for homologous regions of DNA?

Answer 8

• If we have a homologous region of DNA, the DNA repair mechanism within the cell will cause DNA repair through homologous recombination
• This results in the homologous regions swapping and recombining.

Question 9

How does homologous recombination work for exogenous regions of DNA?

Answer 9

-If we put an exogenous piece of DNA in the central part of the homologous region, that homologous recombination mechanism will still work.

Question 10

What frequency does homologous recombination occur at and how is this rare event identified?

Answer 10

-Homologous recombination occurs at low frequency therefore we use the neomycin resistance to use positive drug selection to identify these rare events like the drug G418

Question 11

How do we identify events like random integrations?

Answer 11

-Random integration events can also occur and these can also confer drug resistance. Therefore it is necessary to use negative drug selection to identify these events like HSV-tk, ganciclovir sensitivity

Question 12

Steps involved in gene targeting of embryonic stem cells

Answer 12

• Embryonic stem cells extracted from blastocyst
• Targeting vector introduced by electroporation
• Rare cell carry targeted gene
• Use positive negative selection to identify it
• Then we can grow a pure population of targeted embryonic stem cells

Question 13

What is positive selection?

Answer 13

-Positive selection: Include a gene in the recombined region that confers resistance of those cells to a toxic drug

Question 14

What is negative selection?

Answer 14

-Negative selection: Include a gene in the construct that confers sensitivity to a toxic drug in those cells in which random integration has occurred

Question 15

Positive selection

Answer 15

• Neomycin phosphotransferase
• Makes cells resistant to G418
• Use phosphoglycerate kinase promoter and bovine growth hormone poly A tail

Question 16

Negative selection

Answer 16

• HSV-tk

- Makes cells sensitive to ganciclovir or FIAU