species- Mus musculus Engineering the mouse genome David Ornitz

Transcription

1 species- Mus musculus Engineering the mouse genome David Ornitz

2 Time line for mouse genetic engineering Development of chimeras between embryos with different genotypes Transgenic mice first derived by infecting embryos with retroviruses First DNA injection into mouse eggs 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

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 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

7 Preimplantation mouse development

8 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

9 Routes for Introducing Genes into Mice 1) Microinjection of DNA into zygotes 2) Injection of embryos with recombinant virus 3) Transfection of ES cells with cloned DNA Selection, Characterization Chimera formation Transgenic Mice

10

11 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

12 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

13 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

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

15 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

16

17

18

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

20

21

22

23 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

24 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

25 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.

26 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

27

28 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

29 Gene Knockout critical exon exon exon exon exon genetic engineering using embryonic stem cells exon exon exon exon X

30 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

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

32 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

33 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,

34 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

35 Cell, Vol. 85, 1 4, April 5, 1996, Copyright 1996 by Cell Press Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bhlh Gene MRF4 E. N. Olson,* H.-H. Arnold, P. W. J. Rigby, and B. J. Wold c

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

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

38 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

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

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

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

42 Genomic Editing Zinc finger nucleases (ZFNs) TAL effector nucleases (TALENs) CRISPR/Cas9

43 Genomic Editing General principle is to target a non-specific nuclease (FokI) to a specific DNA sequence Double stranded break will induce non-homologous end joining which can disrupt gene function Single stranded breaks (nicks) can induce homologydirected repair with a double or single stranded DNA template

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

45 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

46 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 Obligate heterodimeric FokI nuclease domains ( KK and EL ) - can reduce the formation of unwanted homodimers - may have improved specificities Single stranded cuts (nickases) can be promoted by inactivating the catalytic activity of one monomer of a ZFN or TALEN dimer

47 Nickase design (single strand break) KK and EL are two obligatory heterodimeric FokI variants kk and el are catalytically inactive monomers (D450A mutations) L-KK/R-EL and L-EL/R-KK are active ZFN pairs nickases can be formed, either by pairing L-KK with R-el or L- kk with R-EL Kim et. al., 2012

48 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 Multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome Cong et al., Science 2013; Mali et al, Nature Methods 2013

49 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 at the 3 of the protospacer PAM - Protospaceradjacent motif

50 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.

51 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 at the 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 nuclease-null

52

53 Fgf14 is a non-secreted cytoplasmic protein with no known biochemical or biological function. Fgf14 is expressed in the central nervous system of embryonic and adult mice. ml igl ml igl

54 Wild type locus Fgf14 targeting 1kb Targeted locus Exon2-LacZ 5' 3' 9kb 7.5kb 9kb 4.5kb WT K/O 6.3kb Neo ES clones Δ Neo

55 In situ and northern blot anaylsis of FGF14 expression FGF14 in situ LacZ in situ wt -/- northern blot 28s wt -/- -/+ wt -/- -/+ 18s

56

57 Fgf14 expression patterns in basal ganglia hippocampus and cortex CC DG CPu GP SN

58

59 Fgf14-/- WT

60 In vivo foot printing Fgf14-/- WT

61 Accelerating rotorod 200 +/ /- -/

62 Sensory Motor Tests

63 Missense m utation in the hum an FGF1 4 gene Van Swieten, J. C., Brusse, E., De Graaf, B. M., Krieger, E., Van De Graaf, R., De Koning, I., Maat-Kievit, A., Leegwater, P., Dooijes, D., Oostra, B. A., and Heutink, P. (2003) A mutation in the fibroblast growth factor 14 gene is associated with autosomal dominant cerebellar ataxia, Am J Hum Genet 72, Dalski, A., Atici, J., Kreuz, F. R., Hellenbroich, Y., Schwinger, E., and Zuhlke, C. (2005) Mutation analysis in the fibroblast growth factor 14 gene: frameshift mutation and polymorphisms in patients with inherited ataxias, Eur J Hum Genet 13, Brusse, E., de Koning, I., Maat-Kievit, A., Oostra, B. A., Heutink, P., and van Swieten, J. C. (2005) Spinocerebellar ataxia associated with a mutation in the fibroblast growth fact 14 gene (SCA27): A new phenotype, Mov Disord 21,

64 Missense mutation in the human FGF14 gene Large, three-generation Dutch family Childhood-onset postural tremor Slowly progressive cerebellar ataxia Only moderate cerebellar atrophy in older patients Dyskinesia Low IQ and deficits in memory Autosomal dominant Mutation in the FGF14 gene T1-weighted MRI (van Swieten et al, Am. J. Hum. Genet., 72: , 2003)

65 Qing Wang JL Lou Maolei Xiao Marie Kozel Fernanda Laezza Benjamin Gerber Kelvin Yamada Jeanne Nerbonne Behavioral Tests Mark Bardgett David Wozniak Behavior Core Dept. of Psychiatry

66