How to construct transgenic mice

How to construct transgenic mice Sandra Beer-Hammer Autumn School 2012 Bad Schandau Pharmakologie und Experimentelle Therapie (APET)

Overview History Generation of embryonic stem (ES) cell lines Generation of knock-out mice Generation of transgenic mice ENU-mutagenesis Seite 2

Time line: from pluripotent cells From Nat Rev Mol Cell Biol Vol. 12 (2011) Seite 3

Time line: from pluripotent cells to transgenic mice From Nat Rev Mol Cell Biol Vol. 12 (2011) Seite 4

to the Nobel Prize for Medicine 2007 Nature 336, 348 (1988) Seite 5

Where do ES cells come from? Seite 6

How to generate ES cell lines? Trophoectoderm Inner Cell Mass Blastocoel Day 5-6 Culture of blastocysts Outgrowth of ICM and trophoblasts Isolation of ICM Day 9 Morphological screening of single colonies and isolation Day 14 ES cell line discard Seite 7

Morphology of blastocysts and trophoblasts Blastocysts Trophoblasts Seite 8

Cell culture of ES cell lines - Cultivation on feeder cells (embryonic fibroblasts) - Cultivation in LIF (Leukemia inhibitory factor) containing medium ES cell culture on fibroblasts Seite 9

Origin of ES cells most ES cell lines from 129 strain (E14, R1 etc) high frequency of teratocarcinomas selection of chimera via skin colour (recipient blastocyst from C57BL/6) strain 129 is agouti (A/A) and light brown (b/b) C57BL/6 is non-agouti (a/a) and black (B/B) chimera are agouti/black germ line transmission: F1 mice (chimxc57bl/6) are agouti (A/a) also ES cell lines from C57BL/6 and BALB/c Seite 10

Methods gene inactivation gene-deficient or knock-out mice additional genetic information transgenic mice knock-in mice Seite 11

Generation of gene deficient mice knock-out Seite 12

Planning a targeting vector 1. 5. December 2002 The mouse genome is mapped Seite 13

Planning a targeting vector Careful planning of all steps, including PCR and Southern Blot strategies Functional inactivation of gene of interest Insertion of a selection marker (neo) into the 1. exon (insertion mutagenesis) Replace 1. exon with selection marker (replacement mutagenesis) Insertion / replacement of essential protein domain (other exon than 1. exon) Seite 14

Classical gene inactivation HSV-TK Bacterial aminoglykosid-phosphotransferase : resistance for G418 (positive selection) HSV-TK Viral thymidine kinase: sensitive to Ganciclovir (negative selection) Seite 15

Generation of gene deficient mice knock-out 2. PCR screening Typical gene targeting experiment: Seite 16

Strategies for screeening: PCR Seite 17

Strategies for screeening: Southern Blot Seite 18

Generation of gene deficient mice knock-out 3. Seite 19

Isolation of Blastocysts day 3.5 after mating Seite 20

Injection of ES Cells Holding pipette Blastocyst (2.5 days) Injection pipette with ES cells Seite 21

Uterus transfer Seite 22

Generation of gene deficient mice knock-out 4. Seite 23

Testing of Germline Transmission Agouti mice carry one allele of the mutated gene E14 mice wt +/- +/- wt kb 17.0 4.0 Chimeric mice are mated with C57BL/6 mice Southern Blot Seite 24

Verification of KO +/+ +/- -/- kb 2.0 LTβR 1.2 GAPDH Northern Blot Western Blot Seite 25

Future of knock-out mice?? Seite 26

Conditional Gene-Targeting Introduction of point mutations Gene replacement neo genetically modified ES cell Tissue-specific knock-outs Inducible knock-outs transient Cre (Frt, Flrt) Expression Seite 27

Conditional Gene-Targeting: 1. Introduction of point mutations Seite 28

Conditional Gene-Targeting: 2. Gene replacement Seite 29

Conditional Gene-Targeting: 3. Tissue-specific knock-out Seite 30

Conditional Gene-Targeting: 3. Tissue-specific knock-out Seite 31

Conditional Gene-Targeting: Combination Cre/Flp Seite 32

Conditional Gene-Targeting: 4. Inducible knock-out Seite 33

Conditional Gene-Targeting: 4. Inducible knock-out Seite 34

Conditional Gene-Targeting: 4. Inducible knock-out Seite 35

Conditional Gene-Targeting: 4. Inducible knock-out Seite 36

Condtional Gene-Targeting: Gene ablation with Diphteria Toxin (DT) Seite 37

The Cre-Zoo (constitutive or inducible) Fluorescent proteins Light-inducible cation channel Cre expression Cre inducible DTR Inducible Cre Inducible gene expression LacZ expression Light-inducible anion channel From Johansson, Genesis 2010 Seite 38

Knock-out versus Transgenic Mice Knock-out/knock-in mice Transgenic mice -Targeted inactivation/mutation - Random integration into the genome of gene in the endogenous locus - Microinjection into pronuclei of oocytes - Introduction of mutation in ES cells via homologous recombination - Expression is dependent on integration locus - Tissue-specific switching on and off - Tissue-specific expression not always assessable Seite 39

Generation of Transgenic Mice Seite 40

The construct: cdna or genomic? cdna often easier to isolate and smaller often less expression with cdna constructs (enhancer/silencer) prokaryotic vector-sequences can inhibit the gene expression no limitations on the length for the microinjection (BACs or even YACs) mostly integration of two transgenes transgene should be differentiated from the endogenous DNA/RNA/protein: - reduction of the 3 not-translated region - insertion of silent point mutations (generation of restriction sites) - insertion of tags (HA, his, myc, strep, etc.) Seite 41

Microinjection of DNA Zona pellucida Injection pipette Holding pipette male pronucleus Embryo (1-cell stadium) female pronucleus nucleolus Seite 42

Practical procedure: transfer of the oocytes vasectomized male were mated with female (plug check) pseudopregnant female oocytes are injected into the oviducts Seite 43

SLy2-transgenic mice T- and B-cell specific promotor (B. Iritani, 1997) Seite 44 Northern Blot Western Blot

Classical transgenic mice frequently used in immunology Examples for antigen-receptor transgenic mice B cells: MD4-anti HEL IgM/IgD transgenic mice T cells: - OT-1: TCR specific for the SIINFEKL peptide of ovalbumine presented on k b - OT-2: TCR specific for chicken ovalbumin 323-339 in the context of I-A b - DO11.10: TCR specific for chicken ovalbumin 323-339 in the context of I-A d not all B-/ T-cells express the transgenic receptor (editing), monoclonal antibodies as anti-idiotypes /anti-clonotypes are available to detect the transgenic B- / and T-cells Seite 45

BAC Transgenic (or knock-out) Mice Seite 46

Find the Right BAC (bacterial artificial chromosome) www.ensembl.org From Johansson, Genesis 2010 Seite 47

BAC Transgenic Mice Vector BAC Purify DNA and inject into pronucleus From Johansson, Genesis 2010 Seite 48

ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 49

ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 50

BAC knock-out (SLy2) Seite 51

BAC knock-out ET Method Overview I 1.) Creating the targeting vector with homologues recombination in E. coli pbad electroporate WI1 2.) 28 C NEO r electroporate pbad WI1 3.) Picking Kana/Chloramph resistant clones / check by PCR + DNA restriction Seite 52

BAC Knock-out ET Method Overview II 4.) pbad electroporate WI1 +NEO 28 C 5.) Thymidine kinase electroporate pbad WI1 +NEO 6.) Picking Amp/Kana/Chloramph resistant clones / check by PCR + DNA restriction Seite 53 Pharmakologie und Experimentelle Therapie (APET)

Verification of Modified BAC Purify DNA and electroporate into ES cells Seite 54

Strategies for mutagenesis in mice γ-irradiation (frequency: 10-50 x 10-5 / Locus) spontaneous mutations (frequency: 5 x 10-6 / Locus) Ethylnitroso-urea (frequency: 150 x 10-5 / Locus) Advantage: single point mutations, high troughput Disadvantage: no molecular marker for cloning Seite 55

ENU-mutagenesis: from the hypothesis. ENU mutagenesis of C57BL/6 mice: 2649 F1, 4584 F3 mice specific phenotype: TNF-production upon stimulation with different TLR-stimuli isolation of peritoneal macrophages under anesthesia (mutants can be breed) Seite 56

.to the identification of a locus (2003). Identification of the LPS2 - mutation Seite 57

to the Nobel prize for medicine 2011 Identification of the LPS2 - gene Seite 58

Thank you! Seite 59