How to construct transgenic mice

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Pharmakologie und Experimentelle Therapie (APET)

How to construct transgenic mice

Sandra Beer-Hammer

Autumn School 2011 Bad Schandau

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Overview

• History

• Generation of embryonic stem (ES) cell lines • Generation of knock-out mice

• Generation of transgenic mice • ENU-mutagenesis

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Cells from ICM differentiate to the embryo

Trophoectoderm

Inner Cell Mass

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How to generate ES cell lines? Culture of blastocysts Outgrowth of ICM and trophoblasts Isolation of ICM Morphological screening

of single colonies and isolation Day 5-6

Day 9

Day 14

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Morphology of blastocysts and trophoblasts

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Cell culture of ES cell lines

ES cell culture on fibroblasts

- Cultivation on feeder cells (embryonic fibroblasts)

- Cultivation in LIF (Leukemia inhibitory factor) containing medium

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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)

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• additional genetic information  transgenic mice

 knock-in mice

Methods

• gene inactivation

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Targeting vector

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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)

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Planning a targeting vector

5. December 2002

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Planning a targeting vector

B cell receptor Known: cDNA

sequence (full-length)

Gene Blast: Search for homologous genomic sequence around 10 min later…..

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Classical gene inactivation

HSV-TK

Bacterial aminoglykosid-phosphotransferase : resistance for G418 (positive selection)

Viral thymidine kinase:

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Generation of gene deficient mice „knock-out“

2.

Typical gene targeting experiment:

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Generation of gene deficient mice „knock-out“

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Isolation of Blastocysts

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

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Generation of gene deficient mice „knock-out“ 4.

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Testing of Germline Transmission E14 mice kB 17.0 4.0 w t w t + / - + / - Southern Blot

Chimeric mice are mated with

Agouti mice carry one allele of the

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Seite 31 Verification of KO + / + + / - - / - 2.0 1.2 kB LTbR GAPDH

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neo neo

transiente Cre - expression

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Conditional Gene-Targeting

neo neo

genetically modified ES cell

• Introduction of point mutations • Delete regulatory sequences • Gene replacement

• Tissue-specific knock-outs • Inducible knock-outs

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neo neo

transiente Cre - expression

Conditional Gene-Targeting

neo neo

genetically modified ES cell

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Conditional Gene-Targeting:

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Conditional Gene-Targeting: 2. Delete regulatory sequences

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Conditional Gene-Targeting: 3. Gene replacement

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

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

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Conditional Gene-Targeting: Combination Cre/Flp

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Conditional Gene-Targeting: 5. Inducible knock-out

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Condtional Gene-Targeting: Gene ablation

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Conditional Gene-Targeting: Gene ablation

CD19-Cre

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The Cre-Zoo (constitutive or inducible)

From Johansson, Genesis 2010

Fluorescent proteins Cre expression Inducible Cre Cre inducible DTR LacZ expression Light-inducible cation channel Light-inducible anion channel Inducible gene expression

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Knock-out versus Transgenic Mice

Knock-out/knock-in mice

-Targeted inactivation/mutation of gene in the endogenous locus

- Introduction of mutation in ES cells via homologous recombination

- Tissue-specific switching on and off

Transgenic mice

- Random integration into the genome

- Microinjection into pronuclei of oocytes

- Expression is dependent on integration locus

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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)

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

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Practical procedure: transfer of the oocytes

• vasectomized male were mated with female (plug check) • pseudopregnant female

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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.)

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Construction of DNA and RNA Expression Control

T- and B-cell specific promotor

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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 kb

- OT-2: TCR specific for chicken ovalbumin 323-339 in the context of I-Ab

- DO11.10: TCR specific for chicken ovalbumin 323-339 in the context of I-Ad

• 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

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Rosa 26-Locus

- Ubiquitary expressed gene

- Gene product with unknown function - high gene targeting frequence

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Find the Right BAC (bacterial artificial chromosome)

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BAC Transgenic Mice

Purify DNA and inject into pronucleus From Johansson, Genesis 2010

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From Johansson, Genesis 2010

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BAC knock-out – Method Overview I

NEOr

WI1 pBAD

Picking Kana/Chloramph resistant clones / check by PCR + DNA restriction

electroporate

2.)

3.)

Creating the targeting vector with homologues recombination in E. coli

WI1

electroporate

1.)

28°C pBAD

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BAC Knock-out – Method Overview II

pBAD WI1

+NEO

Picking Amp/Kana/Chloramph resistant clones / check by PCR + DNA restriction electroporate 4.) 6.) WI1 +NEO pBAD electroporate 5.) Thymidine kinase 28°C

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Verification of Modified BAC

Purify DNA and

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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

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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)

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….to the identification of a locus (2003)….

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…to the Nobel prize for medicine 2011

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