lecture 37 - sae 5

Question 1

describe somatic cell fusion

Answer 1

gene mapping

Question 2

describe gene therapy

Answer 2

conventional gene therapy
genome editing and ips

Question 3

describe cell hybridization/fusion

Answer 3

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

Question 4

describe hybrids created

Answer 4

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

Question 5

is cell fusion efficient

Answer 5

noooo highly inefficient
selectable marker needed to identify hybrids

Question 6

describe hat medium

Answer 6

hypoxanthine aminopterin thymidine
natural marker selection

Question 7

describe de novo pathway

Answer 7

blocked by aminopterin
noble pathway
major dna precursors
makes newly synthesized dna

Question 8

describe salvage pathway

Answer 8

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

Question 9

define hypoxanthine and thymidine

Answer 9

hypoxanthine = converted to guanine by hypoxanthine guanine phosphoribosyltransferase
thymidine = phoshorylated by thymidine kinase

Question 10

describe fusion of the hprt and tk lines

Answer 10

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

Question 11

describe mapping measles virus receptor in humans - experiment

Answer 11

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

Question 12

describe mapping measles virus receptor in humans - results

Answer 12

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

Question 13

what is gene therapy

Answer 13

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

Question 14

name the 2 types of viral vectors

Answer 14

derived from adenovirus
derived from retrovirus

Question 15

describe viral vector derived from adenovirus

Answer 15

will infect all cells even non dividing ones
vectors will not be integrated in genome = transgene diluted and eventually lost = not permanent but faster

Question 16

describe viral vector derived from retrovirus

Answer 16

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

Question 17

describe somatic gene editing

Answer 17

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

Question 18

describe germline gene editing

Answer 18

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

Question 19

which type of gene editing is more common

Answer 19

somatic gene editing

Question 20

what is somatic cell gene therapy

Answer 20

transfer of a gene in somatic cells

Question 21

what is germline gene therapy

Answer 21

transfer of a gene in all cells of an organism through germline transmission

Question 22

describe in vivo vs ex vivo gene therapy

Answer 22

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

Question 23

what does ada-scid stand for

Answer 23

adenosine deaminase - severe combined immunodeficiency disease
rare autosomal disease of immune system - bubble boy disease, affected individuals have essentially no immune system

Question 24

describe ada-scid

Answer 24

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

Question 25

describe construction of an expression vector - ada-scid

Answer 25

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

Question 26

describe partially curing scid

Answer 26

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

Question 27

what is rp

Answer 27

Retinitis pigmentosa
makes cells in retina break down slowly over times = progressive vision loss - like tunnel vision

Question 28

describe rp disease

Answer 28

number of mutations can result in rp
disease causing mutations can be autosomal dom or recessive or x linked

Question 29

what is treatment for rp

Answer 29

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

Question 30

name 3 issues with conventional gene therapy

Answer 30

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

Question 31

what is the solution to problems with conventional gene therapy

Answer 31

directly edit genomic sequences of stem cells from the patient = differentiate to any cell type so can target many tissues

Question 32

describe tools to target specific sequences in genome

Answer 32

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

Question 33

describe how sick cell can be treated

Answer 33

gene therapies = editing genome
now can treat it

Question 34

what are ips and explains

Answer 34

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

Question 35

describe future treatments

Answer 35

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