1pp IMM3031 MHC Function Reverse Genetics (2016)
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Transcript of 1pp IMM3031 MHC Function Reverse Genetics (2016)
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Studying MHC function by
reverse genetics
IMM 3031
Friday April 15th 20162pm lect.
Theatre M2
A/Prof Robyn Slatteryrob n.slatter monash.edu
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Learning objectives
• Be able to describe the basics of geneticengineering approaches including: – Transgenic manipulation
– Homologous recombination to generate knock-out, knock-in and tissue specific modifications
•
Using ES cells, traditional gene targeting and cre-lox
•
Using CRISPR technology
•
Describe examples of each of thesemanipulations and how these have beenused in immunological research.
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Methods of finding ormaking mutants
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1. Screen organism for naturally occurring and“
interesting”
mutations -phenotypes
2. Treat organisms with (UV light, chemicals,
transgenes) and then screen for interestingmutants -phenotypes
3. Generate transgenics
4. Target mutations to specific genes byhomologous recombination
a) cre/lox technology (1993-2013)b) CRISPR technology (since 2013)
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Methods of finding ormaking mutants
4
1. Screen organism for naturally occurring and“
interesting”
mutations -phenotypes
2. Treat organisms with (UV light, chemicals,
transgenes) and then screen for interestingmutants -phenotypes
3. Generate transgenics
4. Target mutations to specific genes byhomologous recombination
a) cre/lox technology (1993-2013)b) CRISPR technology (since 2013)
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Making transgenic mice
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regulatory region
promoter region)
- “specificity”
Coding region
- governs what the gene
product will be
IntronExon
Gene
or
cDNA
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X
1-cell embryo
at pronuclear stage
holding pipette
Construct
transgene
oviduct transfer
Screen litters for transgenic
mice
X
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jellyfishplant
fish
mice
Adding fluorescent protein
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Slattery et al Nature 345, 724 - 726 (1990);Prevention of diabetes in non-obese diabetic I-Ak transgenic mice
Normal MHC class II
Diabetes No diabetes
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In Summary
•
Transgenic mice give us an extra gene i.e.gain of function
•
How do we develop other mutants? – Complete loss of gene function (knock-outs)
– Change of gene function (knock-ins)
– regulated loss of gene function (tissue-specificknock-outs)
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Methods of finding ormaking mutants
11
1. Screen organism for naturally occurring and“
interesting”
mutations -phenotypes
2. Treat organisms with (UV light, chemicals,
transgenes) and then screen for interestingmutants -phenotypes
3. Generate transgenics
4. Target mutations to specific genes byhomologous recombination
a) ES cells and cre/lox technology (1993-2013) b) CRISPR technology (since 2013)
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Using homologous recombination in
ES cells to generate:
• Gene knock-outs
•
Gene knock ins
•
Tissue specific gene knock-outs
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oviduct transfer
Screen litters for transgenic
mice
transgene transfer by transfection
Embryonic stem
cellsBlastocyst
Inner cell mass
Select and transfer ES cellsTo blastocyst
Look for chimeric mice
Breed mice
Genetically modified ES cells give rise to genetically
modified mice
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homologous recombination to
generate“knock-out” mice
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Gene to be targeted
Targeting vector
Process which basically replaces endogenous (functional) gene with “invitro” manipulated (non-functional) gene.
selectable markers
+-
Gangcyclovir
Neo R= Neomycin Resistance Gene
HSV-TK= Herpes Simplex Virus Thymidine Kinase
HSV-TK Neo R
Toxic
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Diabetes. 1994 Mar;43(3):500-4.
ß2M-deficient NOD mice do not develop
insulitis or diabetes.
Wicker LS, Leiter EH, Todd JA, Renjilian RJ, Peterson E,
Fischer PA, Podolin PL, Zijlstra M, Jaenisch R, Peterson LB.
Use of knock-out mice to prove that MHC
class I is required for the generation of
CD8 T cells and the development ofdiabetes
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homologous recombination to
generate“knock-in
” mice
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Concept can be extended to introduce defined changes to test specificfeature or function.
Gene targeting vector
Altered nucleotide s)
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The use of“tissue-specific knock out mice”
to determine the role to MHC molecules have in T1D
-cell
CD8
CD4 CD8
APC
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“tissue specific knockout” of MHC class I
expression from Islet beta cells
-cell
CD8
CD4 CD8
APC
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“tissue-specific
” knock-out mice
Gene to be targeted
Targeting vector
Gene of interest
Flanked by lox sites
Endogenous gene flanked by lox sites
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Cre mediated recombination at lox sites
cre
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In vivo cre mediated recombination at
lox sites
X
#
issue-specific knock-out mouse
Tissue specific cre
cre
Gene flanked by lox
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Islets from class-I-
-bald
NOD and controls
CD4
CD8
2M+/-
Macrophage
2Mloxcre- 2Mloxcre+
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Islets from class-I-
-bald
NOD and controls
CD4
CD8
2M+/-
Macrophage
2Mloxcre- 2Mloxcre+
NOTE:CD8 T cells are still
present in the islet
milieu despite the
lack of MHC class I
expression on isletbeta cells
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Reduced incidence of diabetes inclass I beta bald
NOD mice
C u
m u l a t i v e d i a b
e t e s
I n c
i d e n c e ( % )
Age (days)
20
15
10
5
0 100 120 140 160 180 200 220 240
floxed 2Ma
HIPcre+ n=39
2M-/-
HIPcre+ n=17
floxed 2Ma
HIPcre- N=27
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Use of “tissue-specific” knock-out mice
demonstrated MHC class I is required on
the target beta cells for CD8 T cells to killthem and cause T1D
Proc Natl Acad Sci U S A. 2003 May 27;100(11):6688-93. Epub2003 May 15.
Beta cell MHC class I is a late requirement for diabetes.
Hamilton-Williams EE, Palmer SE, Charlton B, Slattery RM.
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Class I removed from APCs
-cell
CD8
CD4
CD8
APC
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Can remove class I from
many different subsets
beta-cell
CD8
CD8
CD4
B cell mø
dend
CD4
CD8
APC
s
CD8
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Can remove class I from
many different subsets
beta-cell
CD8
CD8
CD4
B cell mø
dend
CD4
CD8
APC
s
CD8
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Using CRISPR-Cas technology to makegenetic modifications in vitro and in
vivo
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CRISPR-cas
its origin and constituents
•
CRISPR = clustered, regularly interspaced, shortpalindromic repeats
•
CRISPR is a genomic locus in some bacteria andarchaea that funtions as an adaptive immune system
against invading phage or plasmids – Encodes an endonuclease
– Stores snippets of foreign sequence•
Transcribed into RNAs that guide the endonuclease by basecomplementarity to cleave foreign nucleic acids at specific sequences
•
Type II CRISPR systems – Encodes endonuclease Cas9 – Has ‘guide RNA’ to direct Cas9 to virtually any desired
genomic sequence
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CRISPR-cas how does this bacterialimmune system work?
Sontheimer et al, 2010, Nature, 438: 45-46.Jenkins, J., Biotechniques, July 2012
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CRISPR-cas
its origin and constituents
•
CRISPR = clustered, regularly interspaced, shortpalindromic repeats
•
CRISPR is a genomic locus in some bacteria andarchaea that funtions as an adaptive immune system
against invading phage or plasmids – Encodes an endonuclease
– Stores snippets of foreign sequence•
Transcribed into RNAs that guide the endonuclease by basecomplementarity to cleave foreign nucleic acids at specific sequences
•
Type II CRISPR systems – Encodes endonuclease Cas9 – Has ‘guide RNA’ to direct Cas9 to virtually any desired
genomic sequence
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Targeted genome editing
Genomic DNA Site-specific dsDNA
break
Cleaving single-strandedRNA-guided Cas9 protein
Genome specific
crRNA sequence
Matching genomicsequence
Genome specific tracrRNA-crRNAchimera
Modification of targeted genome
Sontheimer et al, 2010, Nature, 438: 45-46. Jenkins, J., Biotechniques,July 2012
CRISPR-casTargeted genome editing
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CRISPR-casa two component system
(critical residues for specificity in red, “seed
sequence”) linked to tracrRNA
- interacts with Cas9 protein
Schematic of a CRISPR/Cas-targeted double-strand break
1.
2. Plasmid encoding Cas9transfected into cell
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CRISPR-casgenome editing options
Double stranded break
noDonor template present?
Gene knockout via non-
homologous end joining
Gene disruption(knockout)
NHEJ:
xError-prone Error-free
Gene correctionor deletion
DNA insertion
yes yes
HDR: HDR:
Precise DNA modification via
homology-directed repair
plasmid donor ssOligo donor
A or
A
T
Precise DNA modification
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CRISPR-casgenome editing options
Double stranded break
noDonor template present?
Gene knockout via non-
homologous end joining
Gene disruption(knockout)
NHEJ:
xError-prone Error-free
Gene correctionor deletion
DNA insertion
yes yes
HDR: HDR:
Precise DNA modification via
homology-directed repair
plasmid donor ssOligo donor
A or
A
T
Precise DNA modification
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NHEJ
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CRISPR-casgenome editing options
Double stranded break
noDonor template present?
Gene knockout via non-
homologous end joining
Gene disruption(knockout)
NHEJ:
xError-prone Error-free
Gene correctionor deletion
DNA insertion
yes yes
HDR: HDR:
Precise DNA modification via
homology-directed repair
plasmid donor ssOligo donor
A or
A
T
Precise DNA modification
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HDR
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Repairing DSBs• Non-homologous end-joining (NHEJ)
• Sticks ends back together
• Error-prone (can create knock out)
• Homology directed repair (HDR)
• >100X more efficient for making mutants than traditional gene targeting
due to:
• DS breaks
•
Direct zygote injection (no need for chimeras)
• High fidelity
•
Requires longer regions of homology (usually >500 bp)
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CRISPR Cas9 can also be
used to activate and repress
genes
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(critical residues for specificity in red, “seed
sequence”) linked to tracrRNA
- interacts with Cas9 protein
Schematic of a CRISPR/Cas-targeted double-strand break
1.
2. Plasmid encoding dCas9 transfected into cell
Using a “catalytically dead”Cas9 CRISPR
Cas9 can also be used to activate and
repress genes
dCas9 Binds but does not cut
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Activation of genes using
CRISPR cas9
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Repression of genes usingCRISPR-cas9
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CRISPR-cas
genome editing can be performed for either in
vitro or in vivo studies
Freezing
reagents
CRISPR Workflows
~ 12 weeks
~ 16 weeks
Select and transfer CRISPRmodified embryos
to foster mother
Cryo vials
Gene SynthesisWorkflow
(eg gene knock-in or modification)
Transfect cellsMutationdetection
assay
Serial dilution stable cellsSelect GFP+
cells
ECACC Cells
Media & Supplements
Escort TM transfection
Plates, tips, buffers
PCR reagents
Oligonucleotides
GenElute TM
I n V i t r o s t u d i e s
Microinjectinto
nucleus
CRISPR mRNA Genotype founder pupsfor targeted gene manipulation
Embryo culture mediaCryopreservant
Extract-N-Amp
Oligonucleotides
TM
PCR reagents
Weeks 0.5 0.5 1.0 2.0 4.0 4.0
Weeks 2.0 1.0 2.0 11.0
Media & supplements
Plates, tips, buffers
Cel-1 assay
Single cellproliferation
Media &
supplementsPlates, tips, buffers
VALIDATION
FACS
Isolate One-cellembryo
I n V i v o s t u d i e s
CRISPR
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CRISPR-cas
its potential over the next few years?
•
Can be used for in vitro disease modeling incombination with differentiated tissue derivedfrom induced stem cells –
to study the significance of specific genetic
contributors to phenotypic features –
to modify certain features of the tissue prior toreplacement therapy
• e.g. beta cells (genetically engineered to be resistant torecurrent autoimmune attack) for transplantation into
diabetic patients• Question…do you think beta cells would survive in
transplanted recipients if engineered to lack MHC class I?
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CRISPR-cas
• Can achieve everything older gene targeting
methods can
• Faster
•
cheaper
• More efficient
•
Can target multiple genomic sites at once
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CRISPR-cas
applications in past 2 years
MouseBacteria
RatRice PlantsFish
Drosophila
C. elegans
Yeast
Arabidopsis
Tobacco Plants
AND
non-human primates
Human cells
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•
https://www.youtube.com/watch?
v=2pp17E4E-O8
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See Janeway 8th ed (Immunologists toolbox)Hamilton Williams et al (2003) Beta cell MHC
class I is a late requirement for diabetes PNAS
Vol100(11):6688-6693
deJersey et al (2006) Beta cells cannot directly
prime diabetogenic CD8 T cells in NOD micePNAS Vol104(4):1295-300
Gaj T et al (2013) ZFN, TALEN and CRISPR/Cas-
based methods for genome engineering Trends in
Biotchnology Vol 31(7): pp397-404
Bondy-Donomy & Davidson (2014) To acquire or
resist: the complex biological effects of CRISPR-
Cas systems Trends in Microbiology Vol 22(4):
pp218-225
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