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Biol 483 > Midterm 2A > Flashcards

Midterm 2A Flashcards

Lemieux slides - Lect 1 - 4

1
Q

Transfection of genes - requirements

A

target cell
GOI
transfection method
selection method

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2
Q

transfection method categories

A

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

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3
Q

cell fusion transfection

A
  • 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)
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4
Q

Types of karyon nucleate cells created by cell fusion transfection

A
  • 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)
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5
Q

Chromosome transfer transfection

A
  • 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
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6
Q

Modifying donor cells for chromosome transfer

A

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

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7
Q

Artificial transfection - direct

A
  • 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
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8
Q

Artificial transfection - indirect

A
  • Calcium (Pi) co ppt
  • DEAE + DMSO
  • Liposome carrier
  • Protoplast fusion
  • Electroporation
  • microprojectiles
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9
Q

Calcium + (pi) co ppt

A

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

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10
Q

DEAE + DMSO

A

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

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11
Q

Protoplast fusion

A

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

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12
Q

Viral transduction

A

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)

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13
Q

HAT medium

A

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

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14
Q

Selection premise with mutants

A

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)

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15
Q

mutants selected on HAT

A

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

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16
Q

Other examples of selection post-transfection

A
  • 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)
17
Q

DHFR selection

A

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

18
Q

NeoR selection

A

aminoglycoside phosphotransferase confers resistance to aminoglycoside antimicrobics (eg G418)

typical antimicrobic selection

19
Q

XGPRT selection

A

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

20
Q

purine synthesis flow chart

A

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

21
Q

Senescence quinescence

A

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

22
Q

telomere shortening

A

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)

23
Q

methods of immortilization

A
  • transforming virus (eg SV40)
  • oncogene mutations (extending proliferation)
  • chemical/irradiation mutagenesis (induce tourigenesis by disrupting P53)
  • telomerase (eg hTERT)
24
Q

cons on immortilization

A

unstable genome
changed expression profiles
increased presence of mutations

25
Q

overview of immortilization process

A

1 culture –> immortilization

failure to immortalize –> senescence
success –> cells enter crisis
- escape crisis –> immortal
- may die in crisis

26
Q

SV40

A

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

27
Q

Cos7 cells (define + how to make)

A

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)

28
Q

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

A

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

29
Q

geneitc modifications - before vs now

A

Before: TALENZ/Zn fingers

Now: CRISPR

30
Q

How does mutagenesis work?

A

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

31
Q

TALENS/Znfingers

A

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

32
Q

CRISPR

A

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

33
Q

CRISPR vs TALENS/Znfingers

A

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

34
Q

delivery of crispr into cell

A
  • using a vector construct –> cas9 gene + gDNA
  • separate Cas9 RNA + gRNA
  • cas9 nuclease premade
35
Q

CF1 crispr

A

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

Biol 483 (5 decks)

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