lecture 37 - sae 5 Flashcards
describe somatic cell fusion
gene mapping
describe gene therapy
conventional gene therapy
genome editing and ips
describe cell hybridization/fusion
whole cell fusion
virus binds 2 cells – viral membrane fuses membranes of cells – makes bridge then fused together = now hybrid
high confluence - touch each other
describe hybrids created
hybrids containing genomes from the 2 diff cell lines
genomes of fused cells are unstable = randomly keep or lose chromosomes
hybrid between human and rodent = rodent chromosomes mostly kept and most human chromosomes lost = used to map gene of interest in human
is cell fusion efficient
noooo highly inefficient
selectable marker needed to identify hybrids
describe hat medium
hypoxanthine aminopterin thymidine
natural marker selection
describe de novo pathway
blocked by aminopterin
noble pathway
major dna precursors
makes newly synthesized dna
describe salvage pathway
minor dna precursors
salvage pathway like recycles
can live without as long as major INTACT
requires hprt for dgtp and tk for dttp = 2 genes become essential when major not working
if can provide these precursors then cell will live - even tho major pathway gone
define hypoxanthine and thymidine
hypoxanthine = converted to guanine by hypoxanthine guanine phosphoribosyltransferase
thymidine = phoshorylated by thymidine kinase
describe fusion of the hprt and tk lines
fusion of tk deficient and hprt deficient cell = new fusion of 2 cell lines = kinda like complementation test - so now cell has tk and hprt
now full set of enzymes = survive off of minor pathway
describe mapping measles virus receptor in humans - experiment
measles can only bind to primates not rodents
make hybrids between primate and rodent - some human chroms kept but most lost –> identify cells infected by measles –> determine which human chroms kept in those cells (easy since human chrom structure diff - morphologically diff from rodents)
if receptors for measles lost then no measles
describe mapping measles virus receptor in humans - results
look at all human chroms
3/9 can be affected by measles = look at common number so chrom 1 is common in all that have measles
Probably where receptor for measles encoded
what is gene therapy
involves adding normal wild type copy of a gene to genome of an individual carrying defective mutated copies of the gene - often recessive mutations
finding defective genes
only few of 4000 inherited human diseases are treatable
name the 2 types of viral vectors
derived from adenovirus
derived from retrovirus
describe viral vector derived from adenovirus
will infect all cells even non dividing ones
vectors will not be integrated in genome = transgene diluted and eventually lost = not permanent but faster
describe viral vector derived from retrovirus
many will infect only diving cells but others (hiv) can infect without host cell division
transgene and viral vector will be incorporated into genome of infected cells - more stable
describe somatic gene editing
limited to tissue - targets genes in specific types of cells
edited gene is only contained in target cell type
any changes limited to treated individual - edited gene not passed down to future generations
describe germline gene editing
made early in development so any changes copies into all of new cells = alters genome
copied in every cell = includes sperm and egg
will be passed on to future gens
new - mostly not approved since dont know effects
which type of gene editing is more common
somatic gene editing
what is somatic cell gene therapy
transfer of a gene in somatic cells
what is germline gene therapy
transfer of a gene in all cells of an organism through germline transmission
describe in vivo vs ex vivo gene therapy
in vivo = inject and hope it hits right cells
ex vivo = isolate hematopoietic target cells and alter them and then put them back in person = one can better control and monitor efficiency of gene transfer
what does ada-scid stand for
adenosine deaminase - severe combined immunodeficiency disease
rare autosomal disease of immune system - bubble boy disease, affected individuals have essentially no immune system
describe ada-scid
in absence of ada deoxyadenosine accumulates in t lymphocytes and eventually kills these cells - t lymphocytes responsible for stimulation of antibody producing b cells
first disease treated with gene therapy - 1990
most common treatment = bone marrow transplant
describe construction of an expression vector - ada-scid
sv40 promotor = very strong
wild type gene = put back ada
retroviral vector = allow transgene expresion in human cells
long terminal repeats = needed for vector integration in cells
Neomycin resistance antibiotic marker = needed for selection of human cells that will produce virus in vitro
cannot replicate
describe partially curing scid
isolate wbcs - used vector - put ada genes - grow cells in culture - select and put cells back
worked but in 2007 4/10 patients developed leukemia due to integration of retroviral vectore near proto oncogene (ltr enhances transcription and integrated next to protooncogene - promoter strong)
mostly use adenoviral vector nows = due to potential consequences
what is rp
Retinitis pigmentosa
makes cells in retina break down slowly over times = progressive vision loss - like tunnel vision
describe rp disease
number of mutations can result in rp
disease causing mutations can be autosomal dom or recessive or x linked
what is treatment for rp
5% of rp caused by autosomal recessive mutation of rpe65 retinoid isomerohydrolase
2017 = luxturan approved by fda for treating patients with rpe65 mutations
uses adenoviral vector, in vivo = inject into eye, quite successful
name 3 issues with conventional gene therapy
integration sites cannot be controlled - using retroviral one
expression level of the rescue gene may not be optimal
ex vivo experiment limited to certain cell types
hard to control
what is the solution to problems with conventional gene therapy
directly edit genomic sequences of stem cells from the patient = differentiate to any cell type so can target many tissues
describe tools to target specific sequences in genome
do not need bunch of enzymes
crispr sytem (clustered regularly interspaced short palindromic repeats) uses rna molecule to target specific dna sequences
= to target diff sequences in genome = change crRNA sequences
fix mutated gene into wild type
describe how sick cell can be treated
gene therapies = editing genome
now can treat it
what are ips and explains
induced pluripotent stem cells
if over express diff tfs (proteins) = oct3/4, sox2, c-myc and klf4 = fibroblasts will reprogram and become pluripotent stem cells
can just take fibroblasts from ppl - do not need to isolate stem cells anymore
mature cells can be reprogrammed to become pluripotent - no moral implication of using ips unlike embryonic stem cells
describe future treatments
clinical trials with pluripotent stem cells
isolate somatic cells –> reprogram –> edit using crispr or other methods –> correct gene –> differentiate to cell type of interest –> back to person
not chance of rejection since cells come from humans
