lec 10

Question 1

what causes different cell types?

Answer 1

Differentiation is achieved by the systematic sequential changes in gene expression brought about by interactions between the nucleus and cytoplasm.

Question 2

What is somatic cell nuclear transfer?

Answer 2

reprogramming a cel to a totipotent state by using a nucleus from a somatic cell and cytoplasm from an egg cell.

Question 3

what is the down fall of SCNT?

Answer 3

• SCNT is very inefficient Dolly was born after 277 eggs were used of which 29 were viable and only 3 survived gestation and 1 survived to adulthood.
• ethical issues of using egg cells

Question 4

what did GORDON propose in 2006?

Answer 4

in understanding the processes by which the egg environment turns a Somatic cell pluripotent we can bypass the need for an egg cell.

Question 5

What was discovered in 2006?

Answer 5

Shinya Yamanaka in 2006 came up with a the concept to produce IPSC (induced pluripotent stem cells) in essence they act like personalised embryonic stem cells.
The theory behind it was to find the transcription factors responsible for reprogramming the cell.

Question 6

How were IPSCs made?

Answer 6

This was done by using viral vectors. Because we use viral vectors to insert the TF into the cell use of the IPSCs as autologous replacements might be hindered due to the unknown insertion sites of the TFs. So it increases the risk of tumourgenesis.

Question 7

What was the process in finding the specific TFs

Answer 7

1. Identified 24 TFs in an embryonic stem cell. Only picked TFs as they are master regulators. The experiment was based off the fact that a combination of these may reprogramme the cell.
   
   2. They used a reporter mouse:
      They knew - fbx15 is an embryonic stem cell marker
      They inserted a beta - Geo cassette which is a fusion of B-galactosidase and neomycin resistance genes
      So now we can look for B-galactosidase or neomycin resistance in cells.
      Started off with 24 packaging cells to produce the 24 different viral titres, it was concentrated and used to show that the 24 together will make IPSCs.
   3. Now to find the most effective TFs and the minimum required TFs to induce a pluripotent state was carried out by taking one factor out at a time and seeing if it would reduce colony formation.
   4. It was narrowed down to 10 factors and then 4. The 4 most important IPSC TFs are:
      Oct4, Sox2, Klf4 and cMyc ( the y factors )

Question 8

What are the 4 main factors?

Answer 8

Oct4, Sox2, Klf4 and cMyc

Question 9

What are the ways we check IPSC formation?

Answer 9

• Morphology - flat colonies with tightly packed cells with large nuclei and scant cytoplasm.
  
  • Karyotype analysis - to check for mutations and chromosomal duplications
  • Expression profile- if they express pluripotent markers (oct3/4, sox2, ssea3, tra-1-81)
  • Epigenetic status via methylation analysis - in the promoter regions we get CPG islands which can be methylated or demethylated. We are checking for demethylated regions as this corresponds to pluripotency. Because with methylation it prevents TF binding to the promoter region.
  • In vitro differentiation - if they can differentiate into different germ layers via using medias that will direct the differentiation down different lineages such as (B-tubulin-ectodermal) (Vimentin - mesodermal) (a-feroprotein - endodermal)
    And then do immuno staining to check.
  • Teratomas - just inject into a mouse sub dermally, if there is no niche cells stopping differentiation a teratoma will form expressing all different types of cells from the different germ layers.