Lecture 29

Question 1

What facilitates the cell fusion aspect of hybridization

Answer 1

You add a viral particle because the virus is capable of fusing with the cell (does so in nature, to infect with its genome) so if cells are close enough together that the both fuse with the same viral particle this will create a sort of bridge, facilitating the fusion
Chemicals could also facilitate this, but he talked abt virus more

Question 2

Cell hybridization

Answer 2

Process of fusing two cells together, not necessarily of the same species, they are initially binucleate but that changes after subsequent cell divisions, genome is extremely unstable, chromosomes are often lost during mitosis

Question 3

Human and rodent cell hybridization

Answer 3

The rodent cell tends to be more stable so more of its genetic info tends to be retained
Thought to be because the proteins of the rodent chromosomes just tend to interact better with the whole mitosis thing, but some human genes are still kept and expressed

Question 4

What’s a disadvantage of cell hybridization

Answer 4

Successful fusion rate was low, so there has to be some kind of selection marker to indicate those that were successful vs those that were not

Question 5

What are the pathways for producing new DNA

Answer 5

De novo, which is the most common way, make it from the ground up
Using minor DNA precursors, less common, considered to be a salvage patwhay (note that enzymes required for this patwhay are not essential for cell viability because its a minor patwhay)

Question 6

HPRT

Answer 6

Enzyme in the salvage pathway for DNA synthesis that is in charge of dGTP synthesis, not essential to life because it’s a minor patwhay

Question 7

TK

Answer 7

Enzyme in the salvage pathway for DNA synthesis required for dTTP

Question 8

What selection marker did they choose to isolate the successful fusions

Answer 8

They used a selection medium, the HAT medium, hypoxanthine aminopterin thymidine, aminopterin inhibiting the major pathway of DNA synthesis, so you die, but hypoxanthine and thymidine are the precursors for the salvage pathway, this would rescue cell death
So basically: they use cell lines that are deficient it the enzymes of the salvage patwhay, HPRT or TK, such that when theyre put in the HAT medium tehy die UNLESS they’ve fused with a cell that has the ability to make those proteins
Note: they did a kind of complementation thing where one cell line was HPRT deficient and the other TK deficient, so only by fusion with each other can they survive

Question 9

Measles in humans/primates vs rodents

Answer 9

In human cells theres some kind of cell surface receptor that can bind the measles virus, allowing it to infect us, but not rodents, so theres something different it their receptor/genome that is protective against it

Question 10

How did they identify the gene responsible for the receptor that recognizes measles

Answer 10

They fused primate/human cells with rodent cells, some human DNA was lost because it tends to be more unstable than the rodent genome during this kind of fusion, they put it in HAT medium, looked at which cell could/couldnt bind measles virus, because that would indicate whether the cell possessed the human chromosome containing the gene for that receptor

Question 11

What is gene therapy

Answer 11

Adding a normal copy of the gene to a genome defective in the gene (note works for recessive genes, it isnt a knock out for hyperactivity or anything)

Question 12

In the context of gene therapy, how could you deliver the gene to all cells

Answer 12

Mainly done through viral vectors
Adenovirus: will infect all cells, even nondividing ones, but they will not be integrated into the genome so will eventually be lost
Retrovirus: will affect only dividing cells (but not always, like HIV), these will be incorporated into the genome so it will be more stable and permanent
Obviously not the cancerous versions used, no oncogenes

Question 13

What are the 2 types of gene therapy

Answer 13

Somatic cell: refers to a transfer of a gene in all somatic cells (existing body cells), not passed on
Germline: refers to a transfer of a gene in all cells in an organism because its happening at the germ cell level, passed on

Question 14

In vivo vs ex vivo gene therapy

Answer 14

In vivo meaning in a live patient, injecting it into the tissue
Ex vivo, meaning they remove affected cells, they then expose these cells to the viral vector, allow time for correction, they then re-insert these cells into the patient for improved function (so its like correcting a small portion)
Much better control on ex vivo (you can actually make sure its corrected)

Question 15

ADA-SCIDs

Answer 15

Severe combined immunodeficiency disorder, rare autosomal recessive disease of the immune system
In the absence of ADA (adenosine deaminase), deoxyadenosine accumulates in the T cells, eventually killing them, so theres basically no functional immune system because T cells are required to activate B cells
Is treated with gene therapy (retroviral vector) and bone marrow transplant

Question 16

How specifically did they treat SCIDs with gene therapy

Answer 16

They used a retroviral vector and an ex vivo approach, since the disease primarily affected T cells they isolated the patients WBCs, exposed them to the retroviral vector, once they wild type expression was confirmed they were put back into the patient, giving them some aspect of a functional immune system
Though because T cells arent forever, this isnt a long term fix, requires on going therapys

Question 17

Why do we typically not use gene therapy to treat SCIDs

Answer 17

Because you can’t control where the viral gene will insert itself, and a lot of teh time, 4/10, it embedded itself near a proto onco gene, causing leukemia
The LTR promoter region was leading to over production of the proto onco genes

Question 18

What are the main issues with gene therapy overall

Answer 18

Integration site cannot be determined
Expression level of the rescue gene may not be optimal/enough
Ex vivo experiments is limited to certain types of cells
All of this would be potentially solvable by gene editing

Question 19

Gene editing

Answer 19

Very difficult unitl CRISPR, clustered regularly interspaces short palindromic repeats system
It uses RNA to target specific DNA sequences, this can be adjusted for any sequence by just changing the crRNA,

Question 20

Induced pluripotent stem cells

Answer 20

IPS, so some Japanese people found that if you took a normal fibroblast and overexpressed 4 specific genes it could go back to being a pluripotent stem cell
Theoretically the genome could then be altered (no ethical issues like with an embryo) and then forced to differentiate into a specific tissue type, leading to a cured tissue from the individual itself