Lecture 33 Flashcards
describe creation of fusion cells (3 steps)
- viral particle or chemical creates bridge btwn human and rodent cell
- forms 1 cell with 2 nuclei
- at mitosis, daughter cells each have 1 nucleus with chromosomes from both species
describe genomes of fused cells
what happens in human and rodent hybrids?
UNSTABLE –> randomly keeping or losing chromosomes
rodent chromosomes are predominantly kept, most human chromosomes are lost
why do we need selectable markers?
cell fusion is highly inefficient –> need markers to identify hybrids
what is an example of a natural marker selection
HAT medium
what is HAT?
Hypoxanthine Aminopterin Thymidine
what is the major pathway to make nucleotides?
DE NOVO pathway (from scratch)
what is the minor pathway to make nucleotides?
SALVAGE pathway (recycle)
which pathway for nucleotide production is REQUIRED?
de novo –> can live without salvage pathway
describe the purpose of each component of HAT
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
what are the 2 types of cells required to use HAT medium
- TK-deficient cell
- HPRT-deficient cell
what happens in TK deficient cell with HAT?
aminopterin blocks de novo pathway, and since TK deficient cannot make dTTP –> cells dead
what happens in HPRT deficient cell with HAT?
aminopterin blocks de novo pathway, and since HPRT deficient cannot make dGTP –> cells dead
what happens if theres fusion btwn TK deficient and HPRT deficient cell lines?
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
describe how they mapped the measles virus receptor
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
what is gene therapy?
add WT copy of gene to genome carrying defective/mutated copies of gene
what type of mutations does gene therapy typically treat?
recessive mutations
2 viral vectors for gene therapy
- adenovirus
- retrovirus
describe adenovirus as viral vector (2 characteristics)
- infects ALL cells, even non-dividng ones
- genome expressed as episome, not integrated in genome so will eventually get lost (safer)
describe retrovirus as viral vector (2 characteristics)
- most infect only dividing cells but some can infect without host cell division
- transgene and vector incorporated into genome so more permanent and stable
2 types of gene therapy
- somatic gene editing
- germline gene editing
what is somatic cell gene therapy?
transfer of gene in somatic cells, will not pass down to next gen
what is germline cell gene therapy?
transfer of gene in ALL cells of an organism thru germline transmission and will pass down to next gen
what is in vivo gene therapy?
downside?
inject viral vectors to tissue you want to affect
but less control about which cells get infected
what is ex vivo gene therapy?
benefit?
for hematopoietic system –> isolate blood cell and do gene transfer, then reinfuse into BM
benefit: better control
