mouse genetic engineering - washington university...

Mouse Genetic Engineering

David Ornitz Department of Developmental Biology

Novosibirsk, Russia

Time line for mouse genetic engineering

Development of chimeras between embryos with different genotypes

Genetically modified mice first derived by infecting embryos with retroviruses

First DNA injection into mouse eggs First use of the term “Transgenic”

First embryonic stem cells developed

Germline contribution of ES cells

First genetic modification of an ES cell (HPRT gene)

Improved vectors for homologous recombination

1960s

1974, 1976

1980 1981

1981

1984

1987

1987

Tarkowski, Mintz, Gardner

Jaenisch and Mintz

Gordon, Brinster, Constantini, Lacy, Wagner Martin, Evans, Kaufman

Bradley

Smithies

Thomas and Capecchi.

Time line for mouse genetic engineering - cont.

Phenotypic consequences of targeted genes

Conditional gene targeting-cre/lox

Conditional gene targeting-flip/FRT

Multiple conditional alleles, cre, flip

Somatic cloning of mice

Lentiviral vectors for transgenesis

RNAi in mice

Sleeping Beauty transposon mutagenesis

Conditional Mouse Knockout Project

Genomic editing

1990+

1992/1993

1996

1998-

1998

2002

2002

2005

2006 -

2010 -

Marth, Rajewsky

Dymecki

Martin

Wakayama et al

Lois, Baltimore

Conklin,Rosenquist

Jenkins,Copeland

EUCOMM, KOMP, IMPC

The Nobel Prize in Physiology or Medicine 2007

"for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells"

Mario R. Capecchi Sir Martin J. Evans Oliver Smithies

How do we analyze gene function in mice?

Gene addition (transgenic approach)Permits GOF, DN, and knockdown experimentsEctopic (spatial or temporal) expressionAllows gene regulatory elements to be testedAllows populations of cells to be marked with a reporter gene

Targeted mutationsSpecific genes can be targetedUnexpected phenotypes (lethal phenotype may result prior to the spatial and temporal site of interest)Must be very careful to make a null allele

Tissue-specific (conditional) targeted mutationsProvides some of the best features of gene targeting and transgenic approaches

May be combined with enhancer trap and gene trap experiments.An effective method to circumvent embryonic lethality.

Breeding mice gestation period-19 days

(range is 18-21 days depending on strain) age at weaning-21 dayssexual maturity-females 4-5 weeks, males-6-8 weeksbirthweight-1 gmweaning-8-12 gmadult-30-40 gm

Preimplantation mouse development

Aggregation chimerasBefore the use of microinjection aggregation chimeras were the only way to genetically modify cells and test them during mouse development

Morula aggregation, used to make chimeras between two different genetic backgrounds

ES/EC cell chimeraadd genetically modified cells to a mouse

Routes for Introducing Genes into Mice

1) Microinjection of DNA into zygotes (TALEN, CRISPR)

2) Injection of embryos with recombinant virus

3) Transfection of ES cells with cloned DNA

Transgenic Mice

Selection, Characterization

Chimera formation

Transgenic Mice: Gene addition

Random insertion of DNA into the mouse genome

Permits GOF, DN and knockdown experiments

Allows gene regulatory elements to be tested

Allows populations of cells to be marked with a reporter gene

Occasionally allows endogenous genes to be trapped

Components of a Transgene

promoter + enhancergene coding sequence or cDNApolyadenylation signal

promoter cDNA splice/poly A

Things that are good:introns

Things that are bad:plasmid sequence, lack of introns

example: Elastase Promoter

cell-type specific expression200 bp is sufficient for expression

Pr/En hGH Pr/En splice/poly Apoly A v-rascDNA

Transgenic mouse issues:Tissue specificity

ectopic expression chromosomal integration site may affect expression

Temporal specificityLevel of expressionInsertional mutagenesis

How to make a transgenic mouse1. Fusion Gene Construct 2. Superovulated Female

PromoterATG

Coding Sequence p(A)

Microinjection

3. Germline Integration

Fertilized Eggs

TRANSGENIC MOUSE

4. DNA Analysis

5. Breeding

from Manipulating the Mouse Embryoa laboratory manual, CSHL press

http://mgc.wustl.edu/

http://mgc.wustl.edu

Homologous recombination using embryonic stem cells • First completely unbiased experiment of gene function in an entire mammalian organism.• Discover unanticipated early embryonic roles

Potential problems:

• Embryonic lethality • Redundancy

Events leading to the development of Embryonic Stem Cells

Teratoma

tumors composed of various tissues foreign to their site of origin.

can be formed by transplanting pieces of embryos to extra uterine sites.

Teratocarcinoma

undifferentiated malignant stem cells, metastasize, lethal

made by transplanting day 6-7 mouse embryos under the kidney capsule

resulting tumors can be passaged and cultured to yield embryonal carcinoma cells - EC cells

Embryonic Stem Cells-cont.EC cell linesvariety of stages of differentiation and variable capacity to differentiate

exponential growth and feeder cells are required to prevent differentiation

differentiation can be induced by aggregation

differentiation can be induced by drugs, RA or DMSO.

ES cells

a normal pleuripotent cell line isolated from normal embryo without passing through a tumor stage.

when reintroduced into the embryonic environment ES cells can generate high grade chimeras.

essential to grow on feeder cells (STO fibroblasts or MEFs).

LIF/DIA is required to maintain pleuripotency of ES cells.

Establishment of ES cell lines: transfer intact blastocysts into culturegrow to stage of early post implantation embryodissociate embryonic from extraembryonic tissuecontinue to culture ICM.

2 days afterdisaggregation of ICM

4 days afterdisaggregation

First passage

Chimeric mouse

ES cells derived from 129/SV strain, agouti coat colorinjected into a C57/B6 blastocyst.

Mate chimeric mouse to ‘Black mouse’ (C57/B6J)identify agouti offspring

Gene Knockout

exonexonexonexon

exonexonexonexon

critical exon

X

Gene Knockout

exonexonexonexon

exonexonexonexon

genetic engineering using embryonic stem cells

critical exon

X

Practical issues for basic gene targeting: ✴ length of homology

✴ probes to detect homologous recombination

✴ vector design (with or without negative selection)

Target gene

Targeting vector

Targeted allele

homologous recombinationTarget gene

Targeting vector

Targeted allele

random integration

Homolgous recombination vs. random integration

Issues in interpreting targeted mutationsMust be very careful to make a null allele

haplotype insufficientrecessive

Prove that an allele is nullgene expressionprotein expressionassay for activity of protein

Other types of alleleshypomorphic alleledominant negativelinked random mutation - generate multiple ES linesrecessive

Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P., and Deng, C. (1998). Fibroblast growth factor receptor 2 (FGFR2)-mediated regulation loop between FGF8 and FGF10 is essential for limb induction, Development 125, 753-765.

Arman, E., Haffnerkrausz, R., Chen, Y., Heath, J. K., and Lonai, P. (1998). Targeted disruption of fibroblast growth factor (Fgf) receptor 2 suggests a role for fgf signaling in pregastrulation mammalian development, Proc. Natl. Acad. Sci., U S A 95, 5082-5087.

Issues in interpreting targeted mutations - cont.Neighboring gene effect

✴ PGK promoter - neo may influence a nearby gene

✴ remove the selection cassette to avoid this potential problem

Unexpected phenotype

✴ lethal phenotype may result prior to the developmental stage of interest

Targeted allele

PGK-Neo

Olson EN, Arnold HH, Rigby PW, Wold BJ (1996) Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 85: 1-4.

loxP loxP flox = flanked by lox

Removing the Neo selection cassette

exon

X

exonPGK-NEOexon

exonexonexonexon


Xexonexonexon

critical exon

PGK-NEO

X



exon

X

exonPGK-NEOexon

exonexonexonexon


Xexonexonexon

critical exon

germline promoter - Cre recombinase

PGK-NEO

X



exon

X

exonPGK-NEOexon

exonexonexonexon


Xexonexonexon

critical exon

germline promoter - Cre recombinase

X

Advanced gene targeting issues

Targeting one allele versus both alleles

Gene replacement using recombinases

Knockin mice

Conditional tissue-specific targeted mutations

✴ provides some of the best features of gene targeting and transgenic approaches

✴ may be combined with enhancer trap and gene trap experiments

✴ the targeted gene can be modified using cre and flip recombinases

✴ may be used in conjunction with inducible promoters

exon

critical exon


exon

X

exonexonexon

Xexonexonexon

critical exon

Regulated activation/inactivation of a gene using CreER fusion proteins

exon

critical exon


exon

X

exonexonexon

Xexonexonexon

critical exon


tissue specific promoter -CreER

recombinase Cytosol

exon

critical exon


exon

X

exonexonexon

Xexonexonexon

critical exon



recombinase

+ tamoxifennuclear translocation

Cytosol


exon

X

exonexonexon

Xexonexonexon

critical exon



recombinase


Cytosol

EUCOMM gene targeting vector

SA-βgeo-PA PGK -neo

Criticalexon

Frt LoxP

5' homology 3' homology



Criticalexon

Frt LoxP



Criticalexon

Frt LoxP


Cre

null, reporter allele



Criticalexon

Frt LoxP



Criticalexon

Frt LoxP


Cre

null, reporter allele


Criticalexon

Frt LoxP


Flp

conditional allele


Criticalexon

Frt LoxP

5' homology 3' homologySA-T2A-CreER-PA

Zinc finger nucleases (ZFNs)

TAL effector nucleases (TALENs)

CRISPR/Cas9 RNA-guided nuclease (RGNs)

(RGNs)

Genomic Editing

General principle is to target a non-specific nuclease (FokI, Cas9) to a specific DNA sequence

Double stranded break will induce: • Error-prone non-homologous end-joining (NHEJ), which

leads to variable length insertion/deletion mutations (indels)

• Homology-directed repair (HDR), which can be used to introduce precise alterations directed by a homologous DNA template

Genomic Editing

Genomic Editing

Sander JD & Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32(4):347-355.

Modular assembly of individual zinc fingers Left and Right target sequence with 5 nt spacer

Zinc finger nucleases (ZFNs)

Rémy, 2010

L

R

Nonspecific FokI nuclease domain fused to a customizable DNA-binding domain to target a single genomic locus

FokI nuclease functions as a dimer to cleave double stranded DNA - can form unwanted dimers

- off-target mutagenesis is relatively frequent

Engineered TALEN variant exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells

TAL Effector Nucleases (TALENs)

Guilinger JP, et al. (2014) Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 11(4):429-435.

TAL Effector Nucleases (TALENs)

GATGCATGCACTGTAGTCACTGCA GCT…GTT

TALEN repeats (DNA binding domain)

FokI nuclease domain

FokI nuclease domain

cleavage within

spacer region

DNA target

TALEN repeats (DNA binding domain)

L

R

CRISPR/Cas9 System

CRISPR (clustered regularly interspaced short palindromic repeats)

Streptococcus pyogenes SF370 type II CRISPR locus - 4 genes: Cas9 nuclease two noncoding CRISPR RNAs (crRNAs) trans-activating crRNA (tracrRNA) precursor crRNA (pre-crRNA) array containing nuclease guide

Facilitates RNA-guided site-specific DNA cleavage

Cas9 nucleases can be directed by short guide RNAs (gRNA) to induce precise cleavage at endogenous genomic loci

Cas9 can also be converted into a nicking enzyme

Cong et al., Science 2013; Mali et al, Nature Methods 2013

Multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome

Modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells


CRISPR/Cas9 System cont.

Two components must be introduced into and/or expressed in cells or an organism to perform genome editing:

1. Cas9 nuclease 2. guide RNA (gRNA)

guide RNA: protospacer/crRNA fused to a fixed trans-activating RNA (tracrRNA)

Twenty nucleotides at the 5’ end of the gRNA direct Cas9 to a specific target DNA site using standard RNA-DNA complementarity base-pairing rules

Target sites must lie immediately 5′of a PAM sequence (protospacer adjacent motif) that matches the canonical form 5’-NGG Cas9 nuclease activity can be directed to any DNA sequence of the form N20-NGG simply by altering the first 20 nt of the gRNA to correspond to the target DNA sequence

CRISPR/Cas9 System cont.

Cas9-sgRNA targeting complexes

sgRNA (short guide RNA)

Target recognition and cleavage require protospacer sequence complementary to the spacer and presence of the appropriate NGG PAM sequence 3′ of the protospacer

PAM - Protospacer-adjacent motif

Type II CRISPR specificity suggest that target sites must perfectly match the PAM sequence NGG and the 8- to 12-base “seed sequence” at the 3′ end of the gRNA.

The importance of the remaining 8 to 12 bases is less well understood and may depend on the binding strength of the matching gRNAs or on the inherent tolerance of Cas9 itself.

Mali et al, Nature Methods, 2013

Cas9-sgRNA targeting complexes sgRNA (short guide RNA)

Target recognition and cleavage require protospacer sequence complementary to the spacer and presence of the appropriate NGG PAM sequence 3′ of the protospacer

Cas9 enables programmable localization of dsDNA, RNA, and proteins. Proteins can be targeted to any dsDNA sequence by simply fusing them to Cas9

PAM - Protospacer-adjacent motif

Overview of various Cas9-based applications


Key issues to consider with CRISPR/Cas9 genomic editing technology

Off-target modifications:

Does a given engineered nuclease act at genomic locations other than its intended site?

Critically important because unintended, off-target modifications in cell populations can lead to unexpected functional consequences in both research and therapeutic contexts

- current consensus is that the off-target mutation frequency is relatively low

Tsai SQ, Joung JK (2014) What's Changed with Genome Editing? Cell Stem Cell 15: 3-4.

Lineage tracing using inducible Cre recombinase

Estrogen regulated Cre (CreER)Tetracycline induced Cre expression (TRE-Cre)

Issues:Threshold levels of CRE required to induce recombination.Expression of Cre in multiple lineages or leaky expression.Different reporter mice vary in their sensitivity to CRE.

mTomato

before recombination


mGFPmTomatoROSA26 promoter

mGFPROSA26 promoter


mTomato

before recombination



mGFPROSA26 promoter



recombinase Cytosol



mGFPROSA26 promoter



recombinase


Cytosol

Lgr5-expressing cells give rise to mature taste cells

Lgr5-EGFP-IRES-creERT2, Rosa26-tdTomato

Days after a single tamoxifen induction

Yee et al. Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue. Stem Cells. 2013; 31(5): 992-1000

Lgr5 stem/progenitor cells generate all three types of taste bud cells

Type I taste cells with NTPDase2

Type II taste receptor cells with Trpm5

Type III taste receptor cells with serotonin

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