Mouse Genetic Engineering David Ornitz Department of Developmental Biology

Transcription

1 Mouse Genetic Engineering David Ornitz Department of Developmental Biology Novosibirsk, Russia

2 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, Tarkowski, Mintz, Gardner Jaenisch and Mintz Gordon, Brinster, Constantini, Lacy, Wagner Martin, Evans, Kaufman Bradley Smithies Thomas and Capecchi.

3 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 / Marth, Rajewsky Dymecki Martin Wakayama et al Lois, Baltimore Conklin,Rosenquist Jenkins,Copeland EUCOMM, KOMP, IMPC

4 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

5 How do we analyze gene function in mice? Gene addition (transgenic approach) Permits GOF, DN, and knockdown experiments Ectopic (spatial or temporal) expression Allows gene regulatory elements to be tested Allows populations of cells to be marked with a reporter gene Targeted mutations Specific genes can be targeted Unexpected 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 mutations Provides 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.

6 How do we analyze gene function in mice? Gene addition (transgenic approach) Permits GOF, DN, and knockdown experiments Ectopic (spatial or temporal) expression Allows gene regulatory elements to be tested Allows populations of cells to be marked with a reporter gene Targeted mutations Specific genes can be targeted Unexpected 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 mutations Provides 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.

7 How do we analyze gene function in mice? Gene addition (transgenic approach) Permits GOF, DN, and knockdown experiments Ectopic (spatial or temporal) expression Allows gene regulatory elements to be tested Allows populations of cells to be marked with a reporter gene Targeted mutations Specific genes can be targeted Unexpected 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 mutations Provides 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.

8 Breeding mice gestation period-19 days (range is days depending on strain) age at weaning-21 days sexual maturity-females 4-5 weeks, males-6-8 weeks birthweight-1 gm weaning-8-12 gm adult gm

9 Preimplantation mouse development

10 Aggregation chimeras Before 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 chimera add genetically modified cells to a mouse

11 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 Selection, Characterization Chimera formation Transgenic Mice

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13 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

14 Components of a Transgene promoter + enhancer gene coding sequence or cdna polyadenylation signal promoter cdna splice/poly A Things that are good: introns Things that are bad: plasmid sequence, lack of introns

15 example: Elastase Promoter cell-type specific expression 200 bp is sufficient for expression Pr/En hgh poly A Pr/En v-ras splice/poly A cdna

16 Transgenic mouse issues: Tissue specificity ectopic expression chromosomal integration site may affect expression Temporal specificity Level of expression Insertional mutagenesis

17 How to make a transgenic mouse 1. Fusion Gene Construct 2. Superovulated Female Promoter ATG Coding Sequence p(a) Fertilized Eggs Microinjection 3. Germline Integration 4. DNA Analysis TRANSGENIC MOUSE 5. Breeding

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21 from Manipulating the Mouse Embryo a laboratory manual, CSHL press

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26 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

27 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

28 Embryonic Stem Cells-cont. EC cell lines variety 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.

29 Establishment of ES cell lines: transfer intact blastocysts into culture grow to stage of early post implantation embryo dissociate embryonic from extraembryonic tissue continue to culture ICM. 2 days after disaggregation of ICM 4 days after disaggregation First passage

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31 Chimeric mouse ES cells derived from 129/SV strain, agouti coat color injected into a C57/B6 blastocyst. Mate chimeric mouse to Black mouse (C57/B6J) identify agouti offspring

32 Gene Knockout critical X

33 Gene Knockout critical genetic engineering using embryonic stem cells X

34 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

35 Homolgous recombination vs. random integration homologous recombination Target gene Targeting vector Targeted allele random integration

36 Issues in interpreting targeted mutations Must be very careful to make a null allele haplotype insufficient recessive Prove that an allele is null gene expression protein expression assay for activity of protein Other types of alleles hypomorphic allele dominant negative linked random mutation - generate multiple ES lines recessive

37 Issues in interpreting targeted mutations Must be very careful to make a null allele haplotype insufficient recessive Prove that an allele is null gene expression protein expression assay for activity of protein Other types of alleles hypomorphic allele dominant negative linked random mutation - generate multiple ES lines recessive

38 Issues in interpreting targeted mutations Must be very careful to make a null allele haplotype insufficient recessive Prove that an allele is null gene expression protein expression assay for activity of protein Other types of alleles hypomorphic allele dominant negative linked random mutation - generate multiple ES lines recessive

39 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, 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,

40 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

41 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.

42 Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X PGK-NEO X

43 Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X PGK-NEO X

44 Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X germline promoter - Cre recombinase PGK-NEO X

45 Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X germline promoter - Cre recombinase X

46 Advanced gene targeting issues Targeting one allele versus both alleles Gene replacement using recombinases Knockin mice

47 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

48 Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox critical X

49 Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox tissue specific promoter -CreER recombinase Cytosol critical X

50 Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox tissue specific + tamoxifen nuclear translocation promoter -CreER recombinase Cytosol critical X

51 Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox tissue specific + tamoxifen nuclear translocation promoter -CreER recombinase Cytosol X

52 EUCOMM gene targeting vector Frt SA-βgeo-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology

53 EUCOMM gene targeting vector Frt SA-βgeo-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology Cre Frt LoxP ogy SA-βgeo-PA null, reporter allele

54 EUCOMM gene targeting vector Frt SA-βgeo-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology Cre Flp Frt SA-βgeo-PA LoxP Critical ogy null, reporter allele 3' conditional allele

55 Frt SA-βgeo-PA SA-T2A-CreER-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology

56 Genomic Editing Zinc finger nucleases (ZFNs) TAL effector nucleases (TALENs) CRISPR/Cas9 RNA-guided nuclease (RGNs) (RGNs)

57 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

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

59 Zinc finger nucleases (ZFNs) Modular assembly of individual zinc fingers Left and Right target sequence with 5 nt spacer R L Rémy, 2010

60 TAL Effector Nucleases (TALENs) Nonspecific FokI nuclease domain fused to a customizable DNAbinding 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 Guilinger JP, et al. (2014) Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 11(4):

61 TAL Effector Nucleases (TALENs) FokI nuclease domain TALEN repeats (DNA binding domain) R DNA target GTAGTCACTGCA GCT GTT GATGCATGCACT L TALEN repeats (DNA binding domain) FokI nuclease domain cleavage within spacer region

62 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

63 CRISPR/Cas9 System cont. 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 Sander JD & Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32(4):

64 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

65 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 - Protospaceradjacent motif

66 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.

67 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 PAM - Protospaceradjacent motif Cas9 enables programmable localization of dsdna, RNA, and proteins. Proteins can be targeted to any dsdna sequence by simply fusing them to Cas9

68 Overview of various Cas9-based applications Sander JD & Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32(4):

69 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.

70 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.

71 Regulated activation/inactivation of a gene using CreER fusion proteins ROSA26 promoter mtomato mgfp loxp loxp flox = flanked by lox ROSA26 promoter before recombination mtomato mgfp

72 Regulated activation/inactivation of a gene using CreER fusion proteins ROSA26 promoter mtomato mgfp loxp loxp flox = flanked by lox ROSA26 promoter tissue specific promoter -CreER recombinase before recombination mtomato mgfp Cytosol

73 Regulated activation/inactivation of a gene using CreER fusion proteins ROSA26 promoter mtomato mgfp loxp loxp flox = flanked by lox tissue specific + tamoxifen nuclear translocation promoter -CreER recombinase Cytosol ROSA26 promoter mgfp

74 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):

75 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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