How to construct transgenic mice
Sandra Beer-Hammer
Autumn School 2012 Bad Schandau
Overview
• History
• Generation of embryonic stem (ES) cell lines
• Generation of knock-out mice
• Generation of transgenic mice
to the Nobel Prize for Medicine 2007
How to generate ES cell lines? Culture of blastocysts Outgrowth of ICM and trophoblasts Isolation of ICM Day 5-6 Trophoectoderm
Inner Cell Mass
Blastocoel
Morphological screening
of single colonies and isolation
Day 9
Day 14
Morphology of blastocysts and trophoblasts
Cell culture of ES cell lines
- Cultivation on feeder cells (embryonic fibroblasts)
ES cell culture on fibroblasts
- Cultivation in LIF (Leukemia inhibitory factor) containing medium
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) • 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
Methods
• gene inactivation
gene-deficient or knock-out mice
• additional genetic information
transgenic mice
Planning a targeting vector
1.
5. December 2002
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)
Classical gene inactivation
HSV-TK
HSV-TK
Bacterial aminoglykosid-phosphotransferase : resistance for G418 (positive selection)
Viral thymidine kinase:
Generation of gene deficient mice „knock-out“
2.
Typical gene targeting experiment:
Generation of gene deficient mice „knock-out“
Injection of ES Cells Holding pipette Blastocyst (2.5 days) Injection pipette with ES cells
Generation of gene deficient mice „knock-out“
Testing of Germline Transmission E14 mice kB w t w t + /-+
/-Agouti mice carry one allele of the
mutated gene
kB 17.0
Verification of KO + /+ + /--/ -2.0 kB LTβββRβ 2.0 1.2 LTβββRβ GAPDH
Conditional Gene-Targeting
neo neo
genetically modified ES cell
• Introduction of point mutations
• Gene replacement
• Tissue-specific knock-outs
genetically modified ES cell
• Inducible knock-outs
Conditional Gene-Targeting:
Conditional Gene-Targeting: 2. Gene replacement
Conditional Gene-Targeting: 3. Tissue-specific knock-out
Conditional Gene-Targeting: 3. Tissue-specific knock-out
Conditional Gene-Targeting: Combination Cre/Flp
Conditional Gene-Targeting: 4. Inducible knock-out
Conditional Gene-Targeting: 4. Inducible knock-out
Conditional Gene-Targeting: 4. Inducible knock-out
Conditional Gene-Targeting: 4. Inducible knock-out
Condtional Gene-Targeting:
The Cre-Zoo (constitutive or inducible) Fluorescent proteins Light-inducible cation channel Cre expression Inducible Cre Cre inducible DTR
Knock-out versus Transgenic Mice
Knock-out/knock-in mice
-Targeted inactivation/mutation of gene in the endogenous locus
Transgenic mice
- Random integration into the genome of gene in the endogenous locus
- Introduction of mutation in ES cells via homologous recombination
- Tissue-specific switching on and off
- Microinjection into pronuclei of oocytes
- Expression is dependent on integration locus
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.)
Microinjection of DNA Holding Injection pipette Zona pellucida Holding pipette Embryo (1-cell stadium) male pronucleus female pronucleus nucleolus
Practical procedure: transfer of the oocytes
• vasectomized male were mated with female (plug check) • pseudopregnant female
SLy2-transgenic mice
T- and B-cell specific promotor specific promotor
Classical transgenic mice frequently used in immunology
Examples for antigen-receptor transgenic mice
• B cells: MD4-anti HEL IgM/IgD transgenic mice
• T cells: • 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
Find the Right BAC (bacterial artificial chromosome)
BAC Transgenic Mice
Vector
BAC
BAC knock-out – ET Method Overview I
Creating the targeting vector with homologues recombination in E. coli
WI1 electroporate 1.) 28 28°°CC pBAD NEOr WI1 pBAD electroporate 2.) 28 28°°CC
BAC Knock-out – ET Method Overview II pBAD WI1 +NEO electroporate 4.) 28 28°°CC
Picking Amp/Kana/Chloramph resistant clones / check by PCR + DNA restriction 6.) WI1 +NEO pBAD electroporate 5.) Thymidine kinase 28 28°°CC
Verification of Modified BAC
Strategies for mutagenesis in mice
• γγγγ-irradiation (frequency: 10-50 x 10-5 / Locus)
• spontaneous mutations (frequency: 5 x 10-6 / Locus)
• Ethylnitroso-urea (frequency: 150 x 10-5 / Locus)
• Ethylnitroso-urea (frequency: 150 x 10-5 / Locus) Advantage: single point mutations, high troughput
ENU-mutagenesis: from the hypothesis .
.to the identification of a locus (2003) .
to the Nobel prize for medicine 2011
