How to construct transgenic mice

Transcription

1 How to construct transgenic mice Sandra Beer-Hammer Autumn School 2010 Bad Schandau

2 Methods additional genetic information transgenic mouse line gene inactivation gene-deficient knockout mouse line Jak2 -/- Seite 2

3 Transgenic Mice Seite 3

4 Generation of Transgenic Mice holding pipette cloned gene 1-cell embryos at pronuclear stage injection pipette Seite 4

5 Microinjection of DNA Holding pipette Injection pipette Embryo (1-cell stadium) Seite 5

6 Construction of DNA and Expression Control T- and B-cell specific promotor (B. Iritani, 1997) Seite 6

7 Protein Expression in T and B Cells Seite 7

8 Gene Deficient Mice knock-out Jak2 -/- Seite 8

9 Gene Targeting Method - Overview from Capecchi, 1998 Seite 9

10 Targeting Construct - Design A 17 kb E probe A A X E LTβR: Genomic locus Targeting vector exon kb E neo HSV-TK E probe A neo E E Targeted allele 1 kb Neo: HSV-TK: positive selection negative selection Seite 10

11 Transfection of ES Cells Seite 11

12 Targeting Verification E14 wt +/- kb Southern Blot Seite 12

13 Isolation of Blastocysts (C57/BL6) Seite 13

14 Injection of ES Cells Holding pipette Blastocyst (2.5 days) Injection pipette with ES cells Seite 14

15 Chimeric Offspring Seite 15

16 Testing of Germline Transmission Aguti mice carry one allele of the mutated gene E14 mice wt +/- +/- wt kb Chimeric mice are mated with C57/BL6 mice Southern Blot Seite 16

17 Verification of KO +/+ +/- -/- kb 2.0 LTβR 1.2 GAPDH Northern Blot Western Blot Seite 17

18 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

19 Organ Size and Morphology Impaired Organogenesis of LNs without LTβR Seite 19

20 Immune Cell Subpopulations Reduced DP and SP Thymocytes in SLy1-deficient Mice Seite 20

21 Immune Response in vivo OD 405nm 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 TNP-CGG: IgM KO WT OD 405nm TNP-CGG: IgG1 ** * OD 405nm 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

22 Immune Tolerance H-2 bxd H-2 b Transplant survival (%) 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) days Increased Allograft Survival in SLy1-deficient Mice Seite 22

23 Conditional Gene-Targeting neo genetically modified ES cell Seite 23

24 Procaryotic Rekombinase Systems neo transient Cre (Frt, Flrt) Expression complete / multiple deletions (long distance deletions) conditional gene targeting Seite 24

25 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

26 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

27 BAC Transgenic (or knock-out) Mice Seite 27

28 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

29 Find the Right BAC (bacterial artificial chromosome From Johansson, Genesis 2010 Seite 29

30 BAC Transgenic Mice Purify DNA and inject into pronucleus From Johansson, Genesis 2010 Seite 30

31 ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 31

32 ET cloning versus Recombineering From Johansson, Genesis 2010 Seite 32

33 BAC knock-out Seite 33

34 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

35 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

36 Verification of Modified BAC Purify DNA and electroporate into ES cells Seite 36