1pp imm3031 mhc function reverse genetics (2016)

8/18/2019 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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interesting”






a) cre/lox technology (1993-2013)b) CRISPR technology (since 2013)


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Making transgenic mice

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5

regulatory region

promoter region)

- “specificity”

Coding region

- governs what the gene

product will be

IntronExon

Gene

or

cDNA


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7

X

1-cell embryo

at pronuclear stage

holding pipette

Construct

transgene

oviduct transfer

Screen litters for transgenic

mice

X


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8

jellyfishplant

fish

mice

Adding fluorescent protein


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9

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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interesting”






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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NHEJ:



DNA insertion

yes yes

HDR: HDR:




A or

A

T



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NHEJ


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NHEJ:



DNA insertion

yes yes

HDR: HDR:




A or

A

T



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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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