How to construct transgenic mice

How to construct transgenic mice Sandra Beer-Hammer Autumn School 2011 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 Seite 3

Time line: from pluripotent cells to transgenic mice Seite 4

to the Nobel Prize for Medicine 2007 Seite 5

to the Nobel Prize for Medicine 2007 Seite 6

to the Nobel Prize for Medicine 2007 Seite 7

Where do ES cells come from? Seite 8

Cells from ICM differentiate to the embryo Trophoectoderm Inner Cell Mass Blastocoel Seite 9

How to generate ES cell lines? Culture of blastocysts Day 5-6 Outgrowth of ICM and trophoblasts Isolation of ICM Day 9 Morphological screening of single colonies and isolation Day 14 ES cell line discard Seite 10

Morphology of blastocysts and trophoblasts Blastocysts Trophoblasts Seite 11

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 12

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 13

Methods gene inactivation gene-deficient or knock-out mice additional genetic information transgenic mice knock-in mice Seite 14

Generation of gene deficient mice knock-out Seite 15

Targeting vector 1. Seite 16

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 17

Planning a targeting vector 5. December 2002 The mouse genome is mapped Seite 18

Planning a targeting vector Known: cdna sequence (full-length) B cell receptor Gene Blast: Search for homologous genomic sequence around 10 min later.. Seite 19

Classical gene inactivation HSV-TK Bacterial aminoglykosid-phosphotransferase : resistance for G418 (positive selection) HSV-TK Viral thymidine kinase: sensitive to Ganciclovir (negative selection) Seite 20

Positive and negative selection Seite 21

Generation of gene deficient mice knock-out 2. PCR screening Typical gene targeting experiment: Seite 22

Strategies for screeening: PCR Seite 23

Strategies for screeening: Southern Blot Seite 24

Generation of gene deficient mice knock-out 3. Seite 25

Isolation of Blastocysts day 3.5 after mating Seite 26

Injection of ES Cells Holding pipette Blastocyst (2.5 days) Injection pipette with ES cells Seite 27

Uterus transfer Seite 28

Generation of gene deficient mice knock-out 4. Seite 29

Testing of Germline Transmission E14 mice w t w t + / - + / - Agouti mice carry one allele of the mutated gene kb 17.0 4.0 Chimeric mice are mated with C57BL/6 mice Southern Blot Seite 30

+ / + + / - - / - Verification of KO kb 2.0 LTbR 1.2 GAPDH Northern Blot Western Blot Seite 31

Future of knock-out mice?? Seite 32

Conditional Gene-Targeting Introduction of point mutations Delete regulatory sequences neo genetically modified ES cell te Cre - expression Gene replacement Tissue-specific knock-outs Inducible knock-outs transient Cre (Frt, Flrt) Expression Seite 33

Conditional Gene-Targeting neo genetically modified ES cell te Cre - expression transient Cre (Frt, Flrt) Expression Seite 34

Conditional Gene-Targeting: 1. Introduction of point mutations Seite 35

Conditional Gene-Targeting: 2. Delete regulatory sequences Which role has the Eµ Enhancer for V(D)J recombination? Seite 36

Conditional Gene-Targeting: 3. Gene replacement Seite 37

Conditional Gene-Targeting: 4. Tissue-specific knock-out Seite 38

Conditional Gene-Targeting: 4. Tissue-specific knock-out Seite 39

Conditional Gene-Targeting: Combination Cre/Flp Seite 40

Conditional Gene-Targeting: 5. Inducible knock-out Seite 41

Conditional Gene-Targeting: 5. Inducible knock-out Seite 42

Conditional Gene-Targeting: 5. Inducible knock-out Seite 43

Conditional Gene-Targeting: 5. Inducible knock-out Seite 44

Condtional Gene-Targeting: Gene ablation Seite 45

Conditional Gene-Targeting: Gene ablation CD19-Cre CD4-Cre Seite 46

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 47

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 48

Generation of Transgenic Mice Seite 49

Practical procedure: isolation of oocytes Cycle of mice: 4 days day/night cycle ovulation: every 4 days, around 5hrs after darkening mating: 1-2 female / male (afternoon) plug check next morning around 50% of mice in cycle: 5-10 oocytes superovulation with gonadotropines: 40-60 oocytes isolation out of the oviduct next day (d2.5) Seite 50

Microinjection of DNA Zona pellucida Injection pipette Holding pipette male pronucleus Embryo (1-cell stadium) female pronucleus nucleolus Seite 51

Practical procedure: transfer of the oocytes vasectomized male were mated with female (plug check) pseudopregnant female oocytes are injected into the oviducts Seite 52

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 53

Construction of DNA and RNA Expression Control T- and B-cell specific promotor (B. Iritani, 1997) Seite 54

Protein Expression in T and B Cells Seite 55

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 56

Rosa 26-Locus - Ubiquitary expressed gene - Gene product with unknown function - high gene targeting frequence Seite 57

Transgenic mice: Various possibilities Seite 58

BAC Transgenic (or knock-out) Mice Seite 59

Find the Right BAC (bacterial artificial chromosome) www.ensembl.org From Johansson, Genesis 2010 Seite 60

BAC Transgenic Mice Purify DNA and inject into pronucleus From Johansson, Genesis 2010 Seite 61

ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 62

ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 63

BAC knock-out Seite 64

BAC knock-out 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 65

BAC Knock-out 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 66 Pharmakologie und Experimentelle Therapie (APET)

Verification of Modified BAC Purify DNA and electroporate into ES cells Seite 67

Two possibilities in genetics Seite 68

Strategies for mutagenesis in mice g-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 69

ENU-mutagenesis: GSF Seite 70

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 71

.to the identification of a locus (2003). Identification of the LPS2 - mutation Seite 72

to the Nobel prize for medicine 2011 Identification of the LPS2 - gene Seite 73

Thank you! Seite 74