Midterm 2A

Question 1

Transfection of genes - requirements

Answer 1

target cell
GOI
transfection method
selection method

Question 2

transfection method categories

Answer 2

Fusion of target cell with donor cell (donor cell contains GOI)
chromosome transfer by microcells
artificial transfection
viral transduction

Question 3

cell fusion transfection

Answer 3

• Fusion of cells –> multinucleated cell –> recombination –> mitosis –> modified mononucleate cell
• chemical fusion –> use fusogens (imperfect, low efficiency)
• physical fusion –> electroporation (higher efficiency)
• Fusogens (eg PEG) –> orient water molecules so thermodyn unfavourable to be btw cells (allow CM fusion)

Question 4

Types of karyon nucleate cells created by cell fusion transfection

Answer 4

• Homokaryon = same cells fused together –> multinucleate cell
• heterokaryon = diff cells fused together –> multinucleate cell
• synkaryon = Mononucleate formed by mitosis of a multinucleated cell (eg hybridoma)

Question 5

Chromosome transfer transfection

Answer 5

• Modify donor cell to contain GOI
• Interrupt mitosis –> induces micronucleation –> small partial genome nuclei
• expose donors to cytochalasin B –> disrupt cytoskeleton
• centrifuge donors –> breaks donors into microcells –> each contains a micronuclei
• PEG fusion of microcells with target cell –> transfection

Question 6

Modifying donor cells for chromosome transfer

Answer 6

used for creating microcells for transfection
Modified to trim genome –> become smaller –> easier to pack into microcells

HAC = human artificial chromosome
MAC = mouse artifical chromosome

Question 7

Artificial transfection - direct

Answer 7

• Microinjection
• Fused zygote but unfused pronuclei –> ingect GOI into male pronucleus
• Zygote host contains 3 copies (1 male, 1 female, 1 transgene)
• Grow offspring –> screen for homozygous for transgenic GOI –> use as transgenic founder

Question 8

Artificial transfection - indirect

Answer 8

• Calcium (Pi) co ppt
• DEAE + DMSO
• Liposome carrier
• Protoplast fusion
• Electroporation
• microprojectiles

Question 9

Calcium + (pi) co ppt

Answer 9

indirect artificial transfection
GOI ppt out of soln –> uptake by cells
Few cell types can actually uptake unsolubilized DNA
LOW efficiency –> need high stringency selection to isolate the few that were transfected

Question 10

DEAE + DMSO

Answer 10

indirect artificial transfection
DEAE (+) neutralizes DNA (-) –> allows crossing through CM
DMSO increases CM permeability –> allow uptake of GOI

Question 11

Protoplast fusion

Answer 11

indirect artifical transfection
Donor BACTERIAL cell with GOI –> lysozymes to remove CW –> protoplast form
PEG fusion with target animal cell
(CW prevents membrane fusion therefore needs removal)

problem - Introduces bacterial elements into animal cell

Question 12

Viral transduction

Answer 12

retroviral transduction - Replace pathogenic genes with GOI –> LTR + rev transcriptase allow integration itno host genome

Simian virus 40 (SV40)
- Integrate GOI into SV40
- Cos7 cells mutated to carry T antigens –> allows transduction of SV40
- Selection
>permissive cells = susceptible therefore viral replication and lysis
> nonpermissive = able to halt SV40 replication. % chance for SV40 to integrate into host genome (desirable)

Question 13

HAT medium

Answer 13

hypoxanthine –> purine synth
aminopterin –> inhibits DHFR in purine (de novo) synth + inhibits thymine de novo synth
thymidine –> nucleoside salvaged for thymidine nt synth

Question 14

Selection premise with mutants

Answer 14

based on fucntional complementation –>< restoring lost functions
mutant hosts –> deficient for a gene
transform GOI wiith selectable recovery gene (SRG)
successful transformation –> SRG uptake –> restore lost function

GOI and SRG –> linked together for transfection OR transfected independantly (ease of manufacturing/purification of product)

Question 15

mutants selected on HAT

Answer 15

Human cells – >lesch-nhyan cells –> HGPRT(-)
myeloma –> HGPRT(-) cells

Aminopterin blocks DE NOVO of thymidine and purines –> only cells capable of salvaging synth survive

HAT media allows selection based on HGPRT and TK
HGPRTase = salvaging hypoxanthine into IMP –> purines
thymidine kinase (TK) = salvaging of thymidine into thymine dTTP

therefore only wt cells (created by restoration of fxn by transfection) will be selected/grow

Question 16

Other examples of selection post-transfection

Answer 16

• DHFR - enz for de novo synth of IMP to form purines
• NeoR gene - encodes aminoglycoside phosphotransferase
• XGPRTase - enz for salvaging xanthine into XMP to form purines (alternative to hypoxanthine salvaing)

Question 17

DHFR selection

Answer 17

transfection using mutant DHFR recovery gene –> resistance to methotrexate inhibitiion

grow cells on methotrexate but NO hypoxanthine
no hypoxanthine –> forces purine de novo
methotrexate presence –> selection for only cells carrying the mutant DHFR copy

Question 18

NeoR selection

Answer 18

aminoglycoside phosphotransferase confers resistance to aminoglycoside antimicrobics (eg G418)

typical antimicrobic selection

Question 19

XGPRT selection

Answer 19

Selection on HAT + mycophenolic acid

aminopterin blocks purine de novo –> force salvaging to survive
mycophenolic acid blocks hypoxanthine salvaging –> forced reliance solely on xanthine salvaging

XGPRT(-) failure to transfect –> dies off
XGPRT(+) successful transfection –> selected for/growth

Question 20

purine synthesis flow chart

Answer 20

raw material -> (DHFR) IMP -> (Mycophen acid) XMP -> purines
hypoxanthine -> (HGPRT) IMP
xanthine -> (XGPRT) XMP

DHFR - blocked by aminopterin + methotrexate
IMP -> XMP blocked by mycophenolic acid –> forces exclusive survival by xanthine salvaging

Question 21

Senescence quinescence

Answer 21

senescence = stoppping proliferation due to telomere shortening –> irreversible
fibroblatss (multipotent stem cells undergo senesc after 50-60 divisions)

quinescence = signal induced stopping of proliferation –> reversible

Question 22

telomere shortening

Answer 22

polymerase needs exposed 3’OH from a primer to work
lagging strand needs many primers –> terminal end of lagging can’t place primer
cleave overhang –> cleave sacrificial telomere repeats

more cell divs over life span –> telomeres shortened –> enter senescence to prevent cleaving unique sequences

life span can be extended by telomerases -> low activity enzymes, may need to introduce transgenic telomerases (ge hTERT)

Question 23

methods of immortilization

Answer 23

• transforming virus (eg SV40)
• oncogene mutations (extending proliferation)
• chemical/irradiation mutagenesis (induce tourigenesis by disrupting P53)
• telomerase (eg hTERT)

Question 24

cons on immortilization

Answer 24

unstable genome
changed expression profiles
increased presence of mutations

Question 25

overview of immortilization process

Answer 25

1 culture --> immortilization failure to immortalize --> senescence success --> cells enter crisis - escape crisis --> immortal - may die in crisis

Question 26

SV40

Answer 26

simian virus 40 carries T antigen --> transfection to host --> disable P53 (immortilizaiton) + allow viral replication GOI replaces T antigen --> still need T antigen --> transfect vector into Cos7 cells --> expression T antigen allows viral replication (consequence of immortalization)--> Cos7 hosts eventually die --> therefore GOI expression is temporary

Question 27

Cos7 cells (define + how to make)

Answer 27

CV1 cel Originated in simians SV40 antigen carrier Since SV40 vector T antigen replaced by GOI --> move T antigen into host cell instead to retain immortalization Transfection into cos cells using - SV40 (will have to remove viral ori to prevent replication/lysis) - Artificially (Ca+Pi coppt w/neoR selection)

Question 28

Alternative to SV40 for GOI expression- Benefit over SV40 + why?

Answer 28

use retrovirus replace pathogenic genes with GOI retain integrase + LTRs integration to genome --> GOI is not kicked out --> long term transient expression

Question 29

geneitc modifications - before vs now

Answer 29

Before: TALENZ/Zn fingers Now: CRISPR

Question 30

How does mutagenesis work?

Answer 30

induce DSBs --> allow natural HDR or NHEJ repair to form indels NHEJ = random indels HDR = can be guided using homologous ssDNA to provide specific indel

Question 31

TALENS/Znfingers

Answer 31

DNA binding doamins specific to flanking target site covalenently linked to FOK1 endonuclease binding so FOK1s overlap same site --> need exact alignment --> +/- 1 nt off = no DSB

Question 32

CRISPR

Answer 32

cas9 nuclease chimeric gRNA (crRNA + trRNA fxns) PAM = actual binding site for cas9

Question 33

CRISPR vs TALENS/Znfingers

Answer 33

talen/zn --> need to specifically engineer binding doamins specific to the each target seq crispr --> only need to change gRNA --> relatively easy to do compare to tal/zn binding domain engineering

Question 34

delivery of crispr into cell

Answer 34

- using a vector construct --> cas9 gene + gDNA - separate Cas9 RNA + gRNA - cas9 nuclease premade

Question 35

CF1 crispr

Answer 35

modified version --> use 2 gRNA + nickase Require nicks close together --> resembles RE cut site --> allow modification by indel nicks too far apart --> repair individual nicks w/o modifications made (decrease off targetting) 2 gRNA binding --> increase specificity