Lecture 33

Question 1

describe creation of fusion cells (3 steps)

Answer 1

1. viral particle or chemical creates bridge btwn human and rodent cell
2. forms 1 cell with 2 nuclei
3. at mitosis, daughter cells each have 1 nucleus with chromosomes from both species

Question 2

describe genomes of fused cells

what happens in human and rodent hybrids?

Answer 2

UNSTABLE –> randomly keeping or losing chromosomes

rodent chromosomes are predominantly kept, most human chromosomes are lost

Question 3

why do we need selectable markers?

Answer 3

cell fusion is highly inefficient –> need markers to identify hybrids

Question 4

what is an example of a natural marker selection

Answer 4

HAT medium

Question 5

what is HAT?

Answer 5

Hypoxanthine Aminopterin Thymidine

Question 6

what is the major pathway to make nucleotides?

Answer 6

DE NOVO pathway (from scratch)

Question 7

what is the minor pathway to make nucleotides?

Answer 7

SALVAGE pathway (recycle)

Question 8

which pathway for nucleotide production is REQUIRED?

Answer 8

de novo –> can live without salvage pathway

Question 9

describe the purpose of each component of HAT

Answer 9

Aminopterin blocks de novo pathway

normally will die with just salvage pathway BUT provide pyrimidine (Hypoxanthine for HPRT) and purine (Thymidine for TK) precursors and cells survive

Question 10

what are the 2 types of cells required to use HAT medium

Answer 10

1. TK-deficient cell
2. HPRT-deficient cell

Question 11

what happens in TK deficient cell with HAT?

Answer 11

aminopterin blocks de novo pathway, and since TK deficient cannot make dTTP –> cells dead

Question 12

what happens in HPRT deficient cell with HAT?

Answer 12

aminopterin blocks de novo pathway, and since HPRT deficient cannot make dGTP –> cells dead

Question 13

what happens if theres fusion btwn TK deficient and HPRT deficient cell lines?

Answer 13

aminopterin blocks de novo pathway but the enzyme deficiencies are complemented by the WT in the other cell –> can make dTTP and dGTP and survive

Question 14

describe how they mapped the measles virus receptor

Answer 14

measles receptor is only on primate cells, not rodent cells

create hybrid btwn primate and rodent and see which can be infected
- if hybrid has chromosome coding for receptor –> INFECTED
- if hybrid does not have chromosome coding for receptor –> NOT infected

determine which chromosome is common btwn hybrid cells that were infected

Question 15

what is gene therapy?

Answer 15

add WT copy of gene to genome carrying defective/mutated copies of gene

Question 16

what type of mutations does gene therapy typically treat?

Answer 16

recessive mutations

Question 17

2 viral vectors for gene therapy

Answer 17

1. adenovirus
2. retrovirus

Question 18

describe adenovirus as viral vector (2 characteristics)

Answer 18

1. infects ALL cells, even non-dividng ones
2. genome expressed as episome, not integrated in genome so will eventually get lost (safer)

Question 19

describe retrovirus as viral vector (2 characteristics)

Answer 19

1. most infect only dividing cells but some can infect without host cell division
2. transgene and vector incorporated into genome so more permanent and stable

Question 20

2 types of gene therapy

Answer 20

1. somatic gene editing
2. germline gene editing

Question 21

what is somatic cell gene therapy?

Answer 21

transfer of gene in somatic cells, will not pass down to next gen

Question 22

what is germline cell gene therapy?

Answer 22

transfer of gene in ALL cells of an organism thru germline transmission and will pass down to next gen

Question 23

what is in vivo gene therapy?

downside?

Answer 23

inject viral vectors to tissue you want to affect

but less control about which cells get infected

Question 24

what is ex vivo gene therapy?

benefit?

Answer 24

for hematopoietic system –> isolate blood cell and do gene transfer, then reinfuse into BM

benefit: better control

Question 25

what does ADA-SCID stand for?

Answer 25

Adenosine DeAminase - Severe Combined Immunodeficiency Disease

Question 26

what happens in ADA-SCID

Answer 26

lose ADA so deoxyadenosine accumulates in T cells and kill them

Question 27

most common treatment of SCID

Answer 27

BM transplant

Question 28

describe an expression vector for SCID gene therapy and its 3 components

Answer 28

WT copy of human ADA with some changes
1. LTR required for integration into host genome
2. SV40 strong promoter to allow expression in human cells
3. NEO-resistance for selection of cells in vitro

Question 29

describe the use of the expression vector for SCID

Answer 29

in retrovirus, the expression vector DNA is inserted into T cells from patient, grown and selected, and then reinfused back into patient

Question 30

what happened to some patients treated with the SCID gene therapy? why?

Answer 30

some developed leukemia –> retrovirual vector inserted near proto-oncogene

Question 31

what is Retinitis Pigmentosa? describe gene therapy to treat

Answer 31

RP makes retina cells slowly break down, causing progressive vision loss
- due to mutations
- 5% is caused by autosomal recessive mutation of RPE65

can use AAV to bring normal RPE65 into retina cells

Question 32

3 issues with conventional gene therapy

Answer 32

1. integration sites cannot be controlled
2. expression level of rescue gene may not be optimal
3. ex vivo experiment is limited to certain types of cells

Question 33

solution to the issues with conventional gene therapy?

Answer 33

directly edit genomic sequences from patient stem cells

Question 34

what is a tool to target specific sequences in genome?

Answer 34

CRISPR

Question 35

what is a type of cell that is helpful for gene therapy?

Answer 35

induce pluripotent stem cells