How to construct transgenic mice Sandra Beer-Hammer Autumn School 2010 Bad Schandau
Methods additional genetic information transgenic mouse line gene inactivation gene-deficient knockout mouse line Jak2 -/- Seite 2
Transgenic Mice Seite 3
Generation of Transgenic Mice holding pipette cloned gene 1-cell embryos at pronuclear stage injection pipette Seite 4
Microinjection of DNA Holding pipette Injection pipette Embryo (1-cell stadium) Seite 5
Construction of DNA and Expression Control T- and B-cell specific promotor (B. Iritani, 1997) Seite 6
Protein Expression in T and B Cells Seite 7
Gene Deficient Mice knock-out Jak2 -/- Seite 8
Gene Targeting Method - Overview from Capecchi, 1998 Seite 9
Targeting Construct - Design A 17 kb E probe A A X E LTβR: Genomic locus Targeting vector exon 1 2 3 4 5 6 7 8 9 10 4 kb E neo HSV-TK E probe A neo E E Targeted allele 1 kb Neo: HSV-TK: positive selection negative selection Seite 10
Transfection of ES Cells Seite 11
Targeting Verification E14 wt +/- kb 17.0 4.0 Southern Blot Seite 12
Isolation of Blastocysts (C57/BL6) Seite 13
Injection of ES Cells Holding pipette Blastocyst (2.5 days) Injection pipette with ES cells Seite 14
Chimeric Offspring Seite 15
Testing of Germline Transmission Aguti mice carry one allele of the mutated gene E14 mice wt +/- +/- wt kb 17.0 4.0 Chimeric mice are mated with C57/BL6 mice Southern Blot Seite 16
Verification of KO +/+ +/- -/- kb 2.0 LTβR 1.2 GAPDH Northern Blot Western Blot Seite 17
Phenotype Analyses - Organ size and morphology - Immune cell subpopulations (frequency, function) - Immune response in vitro (Proliferation, Cytokine production) - Immune response in vivo (Immunisation with Ficoll, CGG) - Defense of infections (Listeria, Toxoplasma, etc.) - Immune tolerance (Transplantation of Skin, Heart, Kidney) - Autoimmunity and Allergy (DTH, Asthma, etc.) - etc. Seite 18
Organ Size and Morphology Impaired Organogenesis of LNs without LTβR Seite 19
Immune Cell Subpopulations Reduced DP and SP Thymocytes in SLy1-deficient Mice Seite 20
Immune Response in vivo OD 405nm 0.6 0.5 0.4 0.3 0.2 0.1 TNP-Ficoll: IgM *** * *** * OD 405nm 0,15 0,12 0,09 0,06 0,03 TNP-Ficoll: IgG2a * ** OD 405nm 0,15 0,12 0,09 0,06 0,03 TNP-Ficoll: IgG3 * d0 d4 d7 d14 d21 d0 d4 d7 d14 d21 d0 d4 d7 d14 d21 OD 405nm 0.4 0.3 0.2 0.1 TNP-CGG: IgM KO WT OD 405nm 0.7 0.6 0.5 0.4 0.3 0.2 0.1 TNP-CGG: IgG1 ** * OD 405nm 0.6 0.5 0.4 0.3 0.2 0.1 TNP-CGG: IgG2a * * d0 d4 d7 d14 d21 d0 d4 d7 d14 d21 d0 d4 d7 d14 d21 Impaired Antibody Production in SLy1-deficient Mice Seite 21
Immune Tolerance H-2 bxd H-2 b Transplant survival (%) 100 80 60 40 20 H-2 b H-2 b, wt (n=5) H-2 bxd H-2 b, wt (n=8) H-2 bxd H-2 b, sly-/- (n=10) 10 20 30 40 50 60 days Increased Allograft Survival in SLy1-deficient Mice Seite 22
Conditional Gene-Targeting neo genetically modified ES cell Seite 23
Procaryotic Rekombinase Systems neo transient Cre (Frt, Flrt) Expression complete / multiple deletions (long distance deletions) conditional gene targeting Seite 24
Cre-mediated Deletion in vivo X Cre Gene-targeted mouse Cre mouse (deleter or tissue/cell-specific) Cre constitutive or inducible deletion of gene of interest Seite 25
The Cre-Zoo (constitutive or inducible) Lck DN T cells CD4 DP T cells FoxP3 Treg cell CD19 B cells Mb1 early B cells CD45 Lymphocytes F4/80 Macrophages, Kupffer cells CD11c DCs Albumin Hepatocytes Tie1/2 Endothelial cells etc. Seite 26
BAC Transgenic (or knock-out) Mice Seite 27
BAC Transgenic (or knock-out) Mice 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 28
Find the Right BAC (bacterial artificial chromosome www.ensembl.org From Johansson, Genesis 2010 Seite 29
BAC Transgenic Mice Purify DNA and inject into pronucleus From Johansson, Genesis 2010 Seite 30
ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 31
ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 32
BAC knock-out Seite 33
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 34
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 35
Verification of Modified BAC Purify DNA and electroporate into ES cells Seite 36