Mouse Genetic Engineering David Ornitz Department of Developmental Biology
|
|
- Shanon Powell
- 6 years ago
- Views:
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
12
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
18
19
20
21 from Manipulating the Mouse Embryo a laboratory manual, CSHL press
22
23
24
25
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
30
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
76
species- Mus musculus Engineering the mouse genome David Ornitz
species- Mus musculus Engineering the mouse genome David Ornitz How do we analyze gene function in mice? Gene addition (transgenic approach) Permits GOF, DN and knockdown experiments Ectopic (spatial or
More informationMouse Engineering Technology. Musculoskeletal Research Center 2016 Summer Educational Series David M. Ornitz Department of Developmental Biology
Mouse Engineering Technology Musculoskeletal Research Center 2016 Summer Educational Series David M. Ornitz Department of Developmental Biology Core service and new technologies Mouse ES core Discussions
More informationspecies- Mus musculus Engineering the mouse genome David Ornitz
species- Mus musculus Engineering the mouse genome David Ornitz Time line for mouse genetic engineering Development of chimeras between embryos with different genotypes Transgenic mice first derived by
More informationMouse Genetics 3/8/17. Outline. History of Mouse Genetics. History of the laboratory mouse. Mouse strains. Gene8c mapping How do we find genes?
3/8/17 Mouse Genetics Heather A Lawson Department of Gene8cs Spring 2017 Outline History of the laboratory mouse Mouse strains Gene8c mapping How do we find genes? Gene8c Engineering How do we analyze gene
More informationBart Williams, PhD Van Andel Research Center
A History of Genome Editing in the Laboratory Implications for Translational Applications Bart Williams, PhD Van Andel Research Center Introduction by Matthew Denenberg, MD DeVos Childrens Hospital Disclosures:
More informationCRISPR Applications: Mouse
CRISPR Applications: Mouse Lin He UC-Berkeley Advantages of mouse as a model organism similar to human Can be genetically manipulated Isogenic and congenic genetic background An accelerated lifespan. Well-characterized
More informationTRANSGENIC ANIMALS. transient. stable. - Two methods to produce transgenic animals:
Only for teaching purposes - not for reproduction or sale CELL TRANSFECTION transient stable TRANSGENIC ANIMALS - Two methods to produce transgenic animals: 1- DNA microinjection 2- embryonic stem cell-mediated
More informationUse of Gene Editing Technologies in Rodents. Carlisle P. Landel, Ph.D.
Use of Gene Editing Technologies in Rodents Carlisle P. Landel, Ph.D. The Mouse as A Model Mammal Small, easy to maintain, fecund Well understood genetics Similarity to humans >90% Availability of inbred
More informationIntroduction and History of Genome Modification. Adam Clore, PhD Director, Synthetic Biology Design
Introduction and History of Genome Modification Adam Clore, PhD Director, Synthetic Biology Design Early Non-site Directed Genome Modification Homologous recombination in yeast TARGET GENE 5 Arm URA3 3
More informationCRISPR/Cas9 Genome Editing: Transfection Methods
CRISPR/ Genome Editing: Transfection Methods For over 20 years Mirus Bio has developed and manufactured high performance transfection products and technologies. That expertise is now being applied to the
More informationTRANSGENIC TECHNOLOGIES: Gene-targeting
TRANSGENIC TECHNOLOGIES: Gene-targeting Reverse Genetics Wild-type Bmp7 -/- Forward Genetics Phenotype Gene or Mutations First Molecular Analysis Second Reverse Genetics Gene Phenotype or Molecular Analysis
More informationExperimental genetics - 2 Partha Roy
Partha Roy Experimental genetics - 2 Making genetically altered animal 1) Gene knock-out k from: a) the entire animal b) selected cell-type/ tissue c) selected cell-type/tissue at certain time 2) Transgenic
More informationTRANSGENIC ANIMALS. -transient transfection of cells -stable transfection of cells. - Two methods to produce transgenic animals:
TRANSGENIC ANIMALS -transient transfection of cells -stable transfection of cells - Two methods to produce transgenic animals: 1- DNA microinjection - random insertion 2- embryonic stem cell-mediated gene
More informationGenome editing. Knock-ins
Genome editing Knock-ins Experiment design? Should we even do it? In mouse or rat, the HR-mediated knock-in of homologous fragments derived from a donor vector functions well. However, HR-dependent knock-in
More informationTheoretical cloning project
Theoretical cloning project Needed to get credits Make it up yourself, don't copy Possible to do in groups of 2-4 students If you need help or an idea, ask! If you have no idea what to clone, I can give
More informationEasi CRISPR for conditional and insertional alleles
Easi CRISPR for conditional and insertional alleles C.B Gurumurthy, University Of Nebraska Medical Center Omaha, NE cgurumurthy@unmc.edu Types of Genome edits Gene disruption/inactivation Types of Genome
More informationA Guide to CRISPR/Cas9
Genome editing and beyond freepik A Guide to CRISPR/Cas9 The latest advance in genomic DNA editing is the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system. This simple-touse
More informationCRISPR cas : Presented By: Pooya Rashvand Advised By: Dr. M.Aslanimehr
Journal Club & MSc Seminar CRISPR cas : Presented By: Pooya Rashvand Advised By: Dr. M.Aslanimehr CRISPR - cas : A New tool for Genetic Manipulations from Bacterial Immunity Systems Viral SS DNA RNA Guide
More informationIntroducing new DNA into the genome requires cloning the donor sequence, delivery of the cloned DNA into the cell, and integration into the genome.
Key Terms Chapter 32: Genetic Engineering Cloning describes propagation of a DNA sequence by incorporating it into a hybrid construct that can be replicated in a host cell. A cloning vector is a plasmid
More informationUsing CRISPR for genetic alteration
Using CRISPR for genetic alteration Joffrey Mianné. j.mianne@har.mrc.ac.uk Mary Lyon Centre, MRC Harwell. CRISPR/Cas origins Origin of the CRISPR/Cas system: Clustered-Regularly Interspaced Short Palindromic
More informationNew Plant Breeding Technologies
New Plant Breeding Technologies Ricarda A. Steinbrecher, PhD EcoNexus / ENSSER Berlin, 07 May 2015 r.steinbrecher@econexus.info distributed by EuropaBio What are the NPBTs? *RNAi *Epigenetic alterations
More informationCRISPR: hot, hot, hot
CRISPR: hot, hot, hot 166 CRISPR is the latest technique for genome engineering and is generating tons of excitement due to its versatility, high specificity, and ease of use. CRISPR stands for clustered
More informationLecture 17. Transgenics. Definition Overview Goals Production p , ,
Lecture 17 Reading Lecture 17: p. 251-256, 260-261 & 264-266 Lecture 18: p. 258-264, 508-524 Transgenics Definition Overview Goals Production p.251-256, 260-261, 264-266 315 Definition A transgenic animal
More informationGenome Engineering with ZFNs, TALENs and CRISPR/Cas9
Genome Engineering with ZFNs, TALENs and CRISPR/Cas9 Designer Endonucleases ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced
More informationStudent Learning Outcomes (SLOS) - Advanced Cell Biology
Course objectives The main objective is to develop the ability to critically analyse and interpret the results of the scientific literature and to be able to apply this knowledge to afford new scientific
More informationLecture 8: Transgenic Model Systems and RNAi
Lecture 8: Transgenic Model Systems and RNAi I. Model systems 1. Caenorhabditis elegans Caenorhabditis elegans is a microscopic (~1 mm) nematode (roundworm) that normally lives in soil. It has become one
More informationPLNT2530 (2018) Unit 9. Genome Editing
PLNT2530 (2018) Unit 9 Genome Editing Unless otherwise cited or referenced, all content of this presenataion is licensed under the Creative Commons License Attribution Share-Alike 2.5 Canada 1 Genome Editing
More informationBi Lecture 3 Loss-of-function (Ch. 4A) Monday, April 8, 13
Bi190-2013 Lecture 3 Loss-of-function (Ch. 4A) Infer Gene activity from type of allele Loss-of-Function alleles are Gold Standard If organism deficient in gene A fails to accomplish process B, then gene
More informationGenetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms
Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms No. 1 of 10 1. The mouse gene knockout is based on. (A) Homologous recombination (B) Site-specific recombination
More informationGenome edi3ng with the CRISPR-Cas9 system
CRISPR-Cas9 Genome Edi3ng Bootcamp AHA Council on Func3onal Genomics and Transla3onal Biology Narrated video link: hfps://youtu.be/h18hmftybnq Genome edi3ng with the CRISPR-Cas9 system Kiran Musunuru,
More informationExpert information. Types of genetically engineered animals
Expert information Committee for Genetics and Breeding of Laboratory Animals Types of genetically engineered animals Status: June 2017 Authors: Ingrid Renner-Müller, Munich Johannes Schenkel, Heidelberg
More informationTransgenesis. Stable integration of foreign DNA into host genome Foreign DNA is passed to progeny germline transmission
Transgenic Mice Transgenesis Stable integration of foreign DNA into host genome Foreign DNA is passed to progeny germline transmission integrates into all cells including sperm or egg Knockin mice DNA
More informationUser Instructions:Transfection-ready CRISPR/Cas9 Reagents. Target DNA. NHEJ repair pathway. Nucleotide deletion. Nucleotide insertion Gene disruption
User Instructions:Transfection-ready CRISPR/Cas9 Reagents Background Introduction to CRISPR/Cas9 genome editing In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR)
More informationTransgenic Mice. Introduction. Generation of Transgenic Mice. Transgenic Mice: A Unique Tool for the Study of Mammalian Biology.
Transgenic Mice Charles Babinet, Institut Pasteur, Paris, France Transgenic mice carry exogenous genetic material introduced by the experimenter. Homologous recombination is used to introduce programmed
More informationReturn to Web Version
Page 1 of 7 Page 1 of 7 Return to Web Version ZFN Technology Biowire Volume 10 Article 1 Have your genomic work cut out for you The genomes of several organisms, including humans, have been sequenced,
More informationCRISPR/Cas9 Gene Editing Tools
CRISPR/Cas9 Gene Editing Tools - Separations Simply Spectacular INDELS Identify indels Determine if one or both copies of your gene have indels The Guide-it Genotype Confirmation Kit: Simple detection
More informationLectures 28 and 29 applications of recombinant technology I. Manipulate gene of interest
Lectures 28 and 29 applications of recombinant technology I. Manipulate gene of interest C A. site-directed mutagenesis A C A T A DNA B. in vitro mutagenesis by PCR T A 1. anneal primer 1 C A 1. fill in
More informationA Survey of Genetic Methods
IBS 8102 Cell, Molecular, and Developmental Biology A Survey of Genetic Methods January 24, 2008 DNA RNA Hybridization ** * radioactive probe reverse transcriptase polymerase chain reaction RT PCR DNA
More informationApplications of Cas9 nickases for genome engineering
application note genome editing Applications of Cas9 nickases for genome engineering Shuqi Yan, Mollie Schubert, Maureen Young, Brian Wang Integrated DNA Technologies, 17 Commercial Park, Coralville, IA,
More informationImproving CRISPR-Cas9 Gene Knockout with a Validated Guide RNA Algorithm
Improving CRISPR-Cas9 Gene Knockout with a Validated Guide RNA Algorithm Anja Smith Director R&D Dharmacon, part of GE Healthcare Imagination at work crrna:tracrrna program Cas9 nuclease Active crrna is
More informationksierzputowska.com Research Title: Using novel TALEN technology to engineer precise mutations in the genome of D. melanogaster
Research Title: Using novel TALEN technology to engineer precise mutations in the genome of D. melanogaster Research plan: Specific aims: 1. To successfully engineer transgenic Drosophila expressing TALENs
More informationCRISPR/Cas9 Gene Editing Tools
CRISPR/Cas9 Gene Editing Tools - Guide-it Products for Successful CRISPR/Cas9 Gene Editing - Why choose Guide-it products? Optimized methods designed for speed and ease of use Complete kits that don t
More informationJohn Gurdon was testing the hypothesis of genomic equivalence or that when cells divide they retain a full genomic compliment.
1. (15 pts) John Gurdon won the 2012 Nobel Prize in Physiology or Medicine for work he did in the 1960 s. What was the major developmental hypothesis he set out to test? What techniques did he development
More informationTRANSGENIC ANIMALS. -transient transfection of cells -stable transfection of cells. - Two methods to produce transgenic animals:
Giovanna Gambarotta- Only for teaching purposes. TRANSGENIC ANIMALS -transient transfection of cells -stable transfection of cells - Two methods to produce transgenic animals: 1- DNA microinjection - random
More informationMouse Transgenic Technologies. Siva, Wai Hung TSANG (PhD), Scientific Officer Animal and Plant Care Facility, HKUST
Mouse Transgenic Technologies Siva, Wai Hung TSANG (PhD), Scientific Officer Animal and Plant Care Facility, HKUST Advancement of Animal Care & Use Programs by Transgenic Services Tailor-made (Transgenesis)
More informationGenome Editing with Programmable Nucleases. Jin-Soo Kim Department of Chemistry Seoul National University
Genome Editing with Programmable Nucleases Jin-Soo Kim Department of Chemistry Seoul National University 1 Method of the Year 2011: Engineered Nucleases RNA-guided Cas9 Endonuclease 3 FokI and the First
More informationThe Use of Genetically-Modified Mouse Models to Study the Actin Cytoskeleton
The Use of Genetically-Modified Mouse Models to Study the Actin Cytoskeleton Anthony Kee (PhD) Cellular and Genetic Medicine Unit School of Medical Sciences (a.kee@unsw.edu.au) 2017 Structure of the Prac
More informationMethods for Reverse genetics References:
Methods for Reverse genetics References: 1. Alonso JM, Ecker JR. Moving forward in reverse: genetic technologies to enable genomewide phenomic screens in Arabidopsis. Nat Rev Genet. 2006 Jul;7(7):524-36.
More informationConcepts and Methods in Developmental Biology
Biology 4361 Developmental Biology Concepts and Methods in Developmental Biology June 16, 2009 Conceptual and Methodological Tools Concepts Genomic equivalence Differential gene expression Differentiation/de-differentiation
More informationCRISPR/Cas9: Tools and Applications for Eukaryotic Genome Editing
CRISPR/Cas9: Tools and Applications for Eukaryotic Genome Editing Fei Ann Ran Broad Institute Cambridge, Massachusetts ran@fas.harvard.edu I will provide some background on the CRISPR/Cas9 technology,
More informationGenome manipulation by homologous recombination in Drosophila Xiaolin Bi and Yikang S. Rong Date received (in revised form): 9th May 2003
Xiaolin Bi is a post doctoral research fellow at the Laboratory of Molecular Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. Yikang S. Rong is the principal
More informationA) (5 points) As the starting step isolate genomic DNA from
GS Final Exam Spring 00 NAME. bub ts is a recessive temperature sensitive mutation in yeast. At º C bub ts cells grow normally, but at º C they die. Use the information below to clone the wild-type BUB
More informationNew methods for gene engineering in animals
New methods for gene engineering in animals Technical Journal Club Special Series on Laboratory Animal Science Caihong Zhu 07.06.2016 Overview I. Definition of gene engineering II. History of gene engineering
More informationGenetics Faculty of Agriculture and Veterinary Medicine. Instructor: Dr. Jihad Abdallah Topic 16: Biotechnology
Genetics 10201232 Faculty of Agriculture and Veterinary Medicine Instructor: Dr. Jihad Abdallah Topic 16: Biotechnology 1 Biotechnology is defined as the technology that involves the use of living organisms
More informationTesting Non-Transgenic CRISPR Technology for Wheat Improvement 13 TH IWGS - TULLN, AUSTRIA
Testing Non-Transgenic CRISPR Technology for Wheat Improvement KALI M BRANDT, HILARY L GUNN, BRETT L BUSCHKE, ADAM HEESACKER, NATHALIA MORET TI, ALEXANDER KARASEV, ROBERT S ZEMETRA 13 TH IWGS - TULLN,
More informationAnalysis of gene function
Genome 371, 22 February 2010, Lecture 12 Analysis of gene function Gene knockouts PHASE TWO: INTERPRETATION I THINK I FOUND A CORNER PIECE. 3 BILLION PIECES Analysis of a disease gene Gene knockout or
More informationNew Plant Breeding Techniques Group 1 Targeted Mutagenesis
WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN New Plant Breeding Techniques Group 1 Targeted Mutagenesis Maria Lusser Joint Research Centre, European
More informationCRISPR: A Simple Tool for Answering Complex Questions
CRISPR: A Simple Tool for Answering Complex Questions www.bcm.edu/cbass c_bass@bcm.edu 713-798-8987 CRISPR-Cas systems CRISPRs: Clustered Regularly Interspaced Short Palindromic Repeats Cas: The accompanying
More informationIMPROVEMENT OF CRISPR GENE EDITING EFFICIENCY AND BEYONDS
IMPROVEMENT OF CRISPR GENE EDITING EFFICIENCY AND BEYONDS YONGLUN LUO (ALUN) ALUN@BIOMED.AU.DK VIB, NOV. 21. 2017 Associate Professor, Department of Biomedicine, Aarhus University, Denmark Executive Director,
More informationCRISPRseek Workshop Design of target-specific guide RNAs in CRISPR-Cas9 genome-editing systems
April 2008 CRISPRseek Workshop Design of target-specific guide RNAs in CRISPR-Cas9 genome-editing systems Lihua Julie Zhu August 1st 2014 Outline Background and Motives CRISPRseek Functionality Dependency
More informationCRISPR/Cas9 Mouse Production
CRISPR/Cas9 Mouse Production Emory Transgenic and Gene Targeting Core http://cores.emory.edu/tmc Tamara Caspary, Ph.D. Scientific Director Teresa Quackenbush --- Lab Operations and Communications Coordinator
More informationThe RRPA knock-in allele was generated by homologous recombination in TC1 ES cells.
Supplemental Materials Materials & Methods Generation of RRPA and RAPA Knock-in Mice The RRPA knock-in allele was generated by homologous recombination in TC1 ES cells. Targeted ES clones in which the
More informationSUPPLEMENTARY INFORMATION
doi:10.1038/nature10163 Supplementary Table 1 Efficiency of vector construction. Process wells recovered efficiency (%) Recombineering* 480 461 96 Intermediate plasmids 461 381 83 Recombineering efficiency
More informationSupplementary Figure 1. Diagram for CATCHA construct. Nature Biotechnology doi: /nbt.3444
Supplementary Figure 1 Diagram for CATCHA construct. Supplementary Figure 2 Representative view of ebony (left) and non-ebony (right) F2 flies from experiments described in Fig. 1c. F0 #1 F0 #2 F0 #3 F0
More informationGenome Editing: Cas9 Stable Cell Lines for CRISPR sgrna Validation, Library Screening, and More. Ed Davis, Ph.D.
TECHNICAL NOTE Genome Editing: Cas9 Stable Cell Lines for CRISPR sgrna Validation, Library Screening, and More Introduction Ed Davis, Ph.D. The CRISPR-Cas9 system has become greatly popular for genome
More informationBarley as a model for cereal engineering and genome editing. Wendy Harwood
Barley as a model for cereal engineering and genome editing Wendy Harwood MonoGram 29 th April 2015 www.bract.org BRACT Transformation Platform Over-expression of single genes RNAi based silencing Promoter
More informationGeneration and Application of Genetically Modified Mouse Models of Human Disease.
Generation and Application of Genetically Modified Mouse Models of Human Disease. Nina Balthasar RCUK and BHF Research Fellow Department of Physiology and Pharmacology University of Bristol The Plan Techniques
More informationKEY Reproductive cloning Therapeutic cloning
1. (20 pts) Define Reproductive and Therapeutic cloning. Make sure your descriptions clearly distinguish the critical differences between them. Describe an example of each. Reproductive cloning refers
More informationUnderstanding embryonic head development. ANAT2341 Tennille Sibbritt Embryology Unit Children s Medical Research Institute
Understanding embryonic head development ANAT2341 Tennille Sibbritt Embryology Unit Children s Medical Research Institute Head malformations among the most common category of congenital malformations in
More informationBiology 4361 Developmental Biology Lecture 4. The Genetic Core of Development
Biology 4361 Developmental Biology Lecture 4. The Genetic Core of Development The only way to get from genotype to phenotype is through developmental processes. - Remember the analogy that the zygote contains
More informationHuman Molecular Genetics Assignment 3 (Week 3)
Human Molecular Genetics Assignment 3 (Week 3) Q1. Which one of the following is an effect of a genetic mutation? a. Prevent the synthesis of a normal protein. b. Alters the function of the resulting protein
More informationTransfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine RNAiMAX
Transfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine RNAiMAX INTRODUCTION The CRISPR/Cas genome editing system consists of a single guide RNA
More informationApplications For CRISPR-Cas9 Stable Cell Lines
Applications For CRISPR-Cas9 Stable Cell Lines Presenter: March 22, 2017 Ed Davis, Ph.D. Senior Application Scientist GeneCopoeia, Inc. GeneCopoeia products & services Functional Genomics & Cell Biology
More informationResearch techniques in genetics. Medical genetics, 2017.
Research techniques in genetics Medical genetics, 2017. Techniques in Genetics Cloning (genetic recombination or engineering ) Genome editing tools: - Production of Knock-out and transgenic mice - CRISPR
More informationStem Cel s Key Words:
Stem Cells Key Words: Embryonic stem cells, Adult stem cells, ips cells, self-renewal, differentiation, pluripotent, multipotent, Inner cell mass, Nuclear transfer (Therapeutic cloning), Feeder cells,
More informationTest Bank for Molecular Cell Biology 7th Edition by Lodish
Test Bank for Molecular Cell Biology 7th Edition by Lodish Link download full: http://testbankair.com/download/test-bank-formolecular-cell-biology-7th-edition-by-lodish/ Chapter 5 Molecular Genetic Techniques
More informationIntroduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products
Introduction to CRISPR/Cas9 Background DNA Cleavage and Repair (NHEJ and HDR) Alternative Cas9 Variants Delivery of Cas9 and sgrna Library Products which one is right for you? CRISPR Workflow abm s Toolbox
More informationSupplementary Figures and Figure legends
Supplementary Figures and Figure legends 3 Supplementary Figure 1. Conditional targeting construct for the murine Satb1 locus with a modified FLEX switch. Schematic of the wild type Satb1 locus; the conditional
More informationLentiviral CRISPR guild RNA Cloning Kit for constructing CRISPR targeting grna lentivectors
Lentiviral CRISPR guild RNA Cloning Kit for constructing CRISPR targeting grna lentivectors Cat# Product Name Amount Application grna-h1-gb grna-h1-gp grna-h1-rb grna-h1-rp grna-h1-puro grna-h1-bsd grna-u6-gb
More informationHumanized Cas9 endonuclease expression lentivirus for CRISPR. Cat# Product Name Amounts
Humanized Cas9 endonuclease expression lentivirus for CRISPR Cat# Product Name Amounts LVP681 LVP681-PBS LVP682 LVP682-PBS LVP683 LVP683-PBS LVP678 LVP678-PBS LVP679 LVP679-PBS LVP680 LVP680-PBS LVP707
More information12/31/16. I. Manipulating DNA (9.1) A. Scientists use several techniques to manipulate DNA. 1. DNA is a very large molecule
I. Manipulating DNA (9.1) A. Scientists use several techniques to manipulate DNA 1. DNA is a very large molecule 3. Led to many biotechnology applications- genetic engineering, DNA fingerprinting, cloning,
More informationExam 3 4/25/07. Total of 7 questions, 100 points.
Exam 3 4/25/07 BISC 4A P. Sengupta Total of 7 questions, 100 points. QUESTION 1. Circle the correct answer. Total of 40 points 4 points each. 1. Which of the following is typically attacked by the antibody-mediated
More informationReview Article Is BAC Transgenesis Obsolete? State of the Art in the Era of Designer Nucleases
Journal of Biomedicine and Biotechnology Volume 2012, Article ID 308414, 5 pages doi:10.1155/2012/308414 Review Article Is BAC Transgenesis Obsolete? State of the Art in the Era of Designer Nucleases J.
More informationTrasposable elements: Uses of P elements Problem set B at the end
Trasposable elements: Uses of P elements Problem set B at the end P-elements have revolutionized the way Drosophila geneticists conduct their research. Here, we will discuss just a few of the approaches
More information(i) A trp1 mutant cell took up a plasmid containing the wild type TRP1 gene, which allowed that cell to multiply and form a colony
1. S. pombe is a distant relative of baker s yeast (which you used in quiz section). Wild type S. pombe can grow on plates lacking tryptophan (-trp plates). A mutant has been isolated that cannot grow
More informationBiotech Applications Nucleic acid therapeutics, Antibiotics, Transgenics. BIT 220 End of Chapter 22 (Snustad/Simmons)
Biotech Applications Nucleic acid therapeutics, Antibiotics, Transgenics BIT 220 End of Chapter 22 (Snustad/Simmons) Nucleic Acids as Therapeutic Agents Many diseases (cancer, inflammatory diseases) from
More informationCRISPR GENOMIC SERVICES PRODUCT CATALOG
CRISPR GENOMIC SERVICES PRODUCT CATALOG DESIGN BUILD ANALYZE The experts at Desktop Genetics can help you design, prepare and manufacture all of the components needed for your CRISPR screen. We provide
More informationThe Development and Application of the CRISPR/CAS System as a Powerful New Tool for Genome Editing: A CASe Study. Zoe Dubrow.
The Development and Application of the CRISPR/CAS System as a Powerful New Tool for Genome Editing: A CASe Study Zoe Dubrow Biochemistry 158 1. Introduction Only a hundred and fifty years have passed since
More informationLecture 12-2/14/2001 Transcription factors I
Lecture 12-2/14/2001 Transcription factors I Topics we will cover today transgenic technology (contd from last time) Gene targeting conditional gene targeting gene trapping regulated expression of introduced
More informationSupporting Information
Supporting Information Park et al. 10.1073/pnas.1410555111 5 -TCAAGTCCATCTACATGGCC-3 5 -CAGCTGCCCGGCTACTACTA-3 5 -TGCAGCTGCCCGGCTACTAC-3 5 -AAGCTGGACATCACCTCCCA-3 5 -TGACAGGAACACCTACAAGT-3 5 -AAGGCACCTTTCTGTCTCCA-3
More informationApplicazioni biotecnologiche
Applicazioni biotecnologiche Analisi forense Sintesi di proteine ricombinanti Restriction Fragment Length Polymorphism (RFLP) Polymorphism (more fully genetic polymorphism) refers to the simultaneous occurrence
More informationTITLE: Mammary Specific Expression of Cre Recombinase Under the Control of an Endogenous MMTV LTR: A Conditional Knock-out System
AD Award Number: DAMD17-98-1-8233 TITLE: Mammary Specific Expression of Cre Recombinase Under the Control of an Endogenous MMTV LTR: A Conditional Knock-out System PRINCIPAL INVESTIGATOR: Rama Kudaravalli,
More informationConstruct Design and Cloning Guide for Cas9-triggered homologous recombination
Construct Design and Cloning Guide for Cas9-triggered homologous recombination Written by Dan Dickinson (ddickins@live.unc.edu) and last updated December 2013. Reference: Dickinson DJ, Ward JD, Reiner
More informationGenetics and Genomics in Medicine Chapter 9 Questions
Genetics and Genomics in Medicine Chapter 9 Questions Multiple Choice Questions Question 9.1 Which, if any, of the following can be classified as a type of augmentation therapy? a) Treatment using a small
More informationCRISPR RNA-guided activation of endogenous human genes
CRISPR RNA-guided activation of endogenous human genes Morgan L Maeder, Samantha J Linder, Vincent M Cascio, Yanfang Fu, Quan H Ho, J Keith Joung Supplementary Figure 1 Comparison of VEGF activation induced
More informationChapter 5. Genetic Models. Organization and Expression of Immunoglobulin Genes 3. The two-gene model: Models to Explain Antibody Diversity
Chapter 5 Organization and Expression of Immunoglobulin Genes 3 4 5 6 Genetic Models How to account for: ) Vast diversity of antibody specificities ) Presence of Variable regions at the amino end of Heavy
More informationChapter 5 Genetic Analysis in Cell Biology. (textbook: Molecular Cell Biology 6 ed, Lodish section: )
Chapter 5 Genetic Analysis in Cell Biology (textbook: Molecular Cell Biology 6 ed, Lodish section: 5.1+5.4-5.5) Understanding gene function: relating function, location, and structure of gene products
More informationEfficient generation of conditional knockout mice by CLICK
Efficient generation of conditional knockout mice by CLICK Tomoji Mashimo Genome Editing Research and Development (R&D) Center and Institute of Experimental Animal Sciences, Graduate School of Medicine,
More informationSupplementary Materials
Supplementary Materials Supplementary Methods Supplementary Discussion Supplementary Figure 1 Calculated frequencies of embryo cells bearing bi-allelic alterations. Targeted indel mutations induced by
More information13-1 Changing the Living World
13-1 Changing the Living World In the past, variation was limited to the variations already in nature or random variations that resulted from mutations. Now, scientists can change DNA and swap genes from
More information