Lecture 3: iPSCs

Question 1

Describe DNA

Answer 1

All cells of all organisms store their hereditary info in a double-stranded DNA genetic blueprint.

Question 2

Describe mRNA

Answer 2

Transcribed from one strand of DNA into a complementary sequence. Provides the details on how to assemble (translate into) a protein.

Question 3

Protein

Answer 3

comprised of covalently linked to amino acids. Does much of the work of the cell-structural functions, enzymatic signal.

Question 4

What is the central dogma?

Answer 4

DNA > RNA> Protein

Question 5

What does a promoter do?

Answer 5

It determines what cells will make mRNA from the gene and when.
So every cell type has some promoters that are active and some that are suppressed.
Different sets of promoters are active in pluripotent stem cells than neurons etc. (not the same as the neurons).

Question 6

Explain how we can link an immature neuronal promoter (Doublecortin) to control the expression of GFP

Answer 6

Doublecortin is only generated when neural stem cells are generated.
You can modify the Doublecortin gene to code for a different mRNA sequence (GFP) But still have that new sequence driven by the Doublecortin promoter.

Question 7

___ approaches can be used to improve the generation and purification of neurons.

Answer 7

Molecular

Question 8

What is the FACS machine?

Answer 8

Fluorescent activated cell sorting of reporter lines can improve purity.
This tool works by purifying different subset of cells and splits into different channels.
Use antibody to pull the cells out.

Question 9

Do your neurons have the same DNA as your skin cells.

Answer 9

Yes, every cell in your body has the same set of DNA and genes.
But they are different because of differential gene expression.

Question 10

What makes your neurons different from your skin cells?

Answer 10

Different sets of genes are being expressed by skin cells vs. neurons.
Terminally differentiation (won’t become another cell).

Question 11

___ determine what cells will make what specific mRNAs and when

Answer 11

Promoters
So every cell type has some promoters that are active and some that are suppressed.
Different sets of promoters are active in pluripotent stem cells than skin cells or neurons.
- Transcription factors control genes and different ones exist for different cell types.

Question 12

The ___ promoter is only turned on and only makes Fbx15 mRNA and Fbx15 protein in a _______

Answer 12

Fbx15
pluripotent embryonic stem cells
It is not expressed in any differentiated cells.

Question 13

Describe the modifications that can be done to the Fbx15.

Answer 13

You can modify the Fbx15 gene to code for a different mRNA sequence. But still have that new sequence “driven” by the Fbx15 promoter

Question 14

Describe the switch between the GFP and other drug resistance proteins.

Answer 14

Instead of putting GFP they switched in a gene that codes for a drug resistance protein (Bgeo). The Bgeo breaks down all cells except for specific ones.

Question 15

When you do this what type of cells will make this Bgeo protein?

Answer 15

Only cells that normally express Fbx15 pluripotent stem cells.

Question 16

When you switch in a gene that codes for a drug resistance protein (Bgeo) what type of cells will not die if you treat them with that drug?

Answer 16

Only cells that produce the Bgeo drug resistance gene driven by Fbx15 pluripotent stem cells.

Question 17

Describe what happens if you use viruses to express certain genes in fibroblasts and find that it turns on the Fbx15 promoter.

Answer 17

If you use viruses to express certain genes in fibroblasts and find that it turns on the Fbx15 promoter, then it suggests that those genes have made the fibroblasts more like embryonic stem cells.

Question 18

The Fbx15 genes are only expressed in ____ stem cells

Answer 18

mouse embryonic stem cells not differentiated cells like fibroblasts so they used the Fbx15 promoter. to drive the expression of antibiotic resistance gene (bgeo).

Question 19

How do you get the skin cell?

Answer 19

Used transcription factors in embryonic stem cells and over expressed them.

Question 20

Describe the generation of iPS cells from MEF cultures via ___ factors.

Answer 20

24 factors that are highly expressed in pluripotent stem cells but not stem cells.

Question 21

Describe the induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures defined by factors.

Answer 21

1.You first make Bgeo to kill off other stuff
2. Apply drug
3. Kill off skin cells.
4. Only Fbx15 ones remain.

Question 22

__ factor iPSCs expressed ESC associated mRNAs and can differentiate into all __ germ lineages.

Answer 22

24 factors
3 germ lineages
surviving cells could become any other type of cell.

Question 23

They found __ factors when each was individually removed produced no colonies at day 10

Answer 23

10
When these 10 factors were combined they induced more colonies than the 24 combined factors.

Question 24

There are __ key factors needed

Answer 24

4
C-Myc, Klf4, Sox2, Oct 3/4

Question 25

What types of analyses helped support the narrowing of the growth factors?

Answer 25

Teratoma formation confirmed production of cells from all 3 germ layers.
(Inject cells into mouse with no immune system and showed all 3 germ layers.
Microarray RNA analysis showed very similar Gene expression profiles for iPSCs and ESCs

Question 26

Describe the induction of pluripotent stem cells from adult human fibroblasts defined factors.

Answer 26

It was found that the same factors work in humans as discovered by Takahashi and Yamanaka