Experimental genetics - 2 Partha Roy

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1 Partha Roy Experimental genetics - 2

2 Making genetically altered animal 1) Gene knock-out k from: a) the entire animal b) selected cell-type/ tissue c) selected cell-type/tissue at certain time 2) Transgenic animal

3 Gene knock-out

4 How to create knockout mice Aim : To create a mouse with gene A knockout k Gene A (exons 1,2,3) WT genome Make a gene construct neor neor- neomycin Resistance gene

5 B black coat color Genotype: (A+/A+; B/B) Sperm: A+, B X (A+/A+; B/B) Egg: A+, B Derive stem cells (ES cells) from early embryo (A+/A+; B/B) Introduce expression vector into black ES cells ES: embryonic stem cells

6 Black ES cells (A+/A+, B/B) Gene A 2 3 WT genome: A 3 x2 (copies) Expression vector 1 neor Homologous recombination (exchange of DNA regions) Resultant Copy 1 Copy 2 : unaffected Neo (A+/A-; B/B) heterozygous for A k/o

7 X b white coat color (A+/A+; b/b) Sperm: A+, b Inject black ES cells into early embryo Implant embryo in pseudo-pregnant mouse (A+/A+; b/b) egg: A+, b (A+/A+; b/b) (A+/A-; B/B) B- dominant allele l b- recessive allele Chimeric mice (A+/A+; b/b <>A+/A-; B/B)

8 (A+/A+; b/b); (A+/A-; B/B) male Sperms: 1) A+, b 2) A+/ B 3) A-/B X female Eggs: A+/b (A+/A+; b/b) (A+/A+; b/b) (A+/A+; B/b) (A-/A+; B/b) Heterozygous k/o How do you sort a heterozygous black pup from a pool of black pups???

9 X (A-/A+; B/b) (A-/A+; B/b).... (A-/A-) homozygous k/o Homozygous k/o %: 25% progeny Confirm b? by? LOOK FOR defects

10 Problem: If the gene is essential for development, homozygous embryos die (embryonic lethal) You might also want to k/o gene only in certain cell type or tissue and/or only at certain time So, devise strategies to conditionally knock out gene 1) CRE-LOX system 2) Tetracycline-inducible system

11 Cre-Lox system (cell-type k/o) Cre a bacteriophage recombinase recognizes a pair of loxp binding sites flanking a gene of interest LoxP sequence: 13 bp 8bp 13 bp ATAACTTCGTATA - GCATACAT - TATACGAAGTTAT ATAACTTCGTATA TATTGAAGCATAT palindromic

12 Close-up of Cre-lox recombination 1 2 ATAACTTCGTATA - GCATACAT - TATACGAAGTTAT Cre-mediated recombination 3 4 ATAACTTCGTATA - GCATACAT - TATACGAAGTTAT Products 1 ATAACTTCGTATA - GCATACAT - TATACGAAGTTAT & 3 2 ATAACTTCGTATA - GCATACAT - TATACGAAGTTAT 4

13 Jackson lab

14 Action of Cre on Lox-flanking gene loxp loxp Cre gene recombination 3 4 gene gene Cyclized byproduct (excised) 1 4

15 Genetic Engineering of the vector in Cre-Lox system Gene X (exons 1, 2,3) You are interested in knocking out gene X Vector design Cre Gene X disrupted in the presence of Cre Without Cre, normal expression of gene X

16 Lox P- mouse (X/loxP) X Cre mouse (heterozygous for X-) (X/X-; Cre) Cre Tissue/cell-specific promoter.. (loxp/x-; cre) (normal expression of gene X except in specific cell-type/tissue where Cre is expressed) Assay for defects in the cell/tissue type

17 K/o at specific time (Inducible expression system) ) Drug induced (you add a drug; either turn ON or OFF gene expression) Tetracycline : Tet-OFF : presence of tetracycline REPRESSES transcription OR Tet -ON : presence of tetracycline INITIATES transcription A li ti lt d ll Application: cultured cells, whole animal (feeding water containing doxycycline)

18 Repressor binds to the operator sequence and prevents gene expression TetR TRE P Tet-Repressor X promoter Tetracycline operator sequence (TRE or tetracycline responsive element) NO Gene expression Gene Inducer will displace the repressor from binding to the operator seq. Add Tet TetR Gene expression TRE P Gene Principle of tet-inducible system

19 Modification from the original system TtR TetR Transcriptional repressor (original) TetR VP16 Transactivator (herpes virus) Fusion protein now becomes transcriptional ti ACTIVATOR (tta tetracycline controlled transactivator) VP16 rtetr Fusion protein CANNOT transactivateti t (rtta reverse tta) Point mutated form of TetR

20 Tet-OFF system (uses tta) NO Gene expression TetR TRE P Gene expression Gene TRE TetR P X Gene (no Tet) (add Tet) Tet-ON system (uses rtta) rtetr X NO Gene expression rtetr TtR Gene expression TRE P Gene TRE P Gene (no Tet) (add Tet)

21 Combining Cre-Lox and Tet-ON systems Lox P- mouse (X/loxP) X Cre mouse (heterozygous for X-) (X/X-; Cre, rtta) Cre TRE Tissue/cell-specific promoter. (loxp/x-; Cre, rtta). Normal expression of gene X unless Cre is expressed in specific cell-type under the influence of tetracycline (gene is knocked out)

22 Making homozygous k/o is not easy and sometimes not possible Alternative strategy: Introduce a dominant negative mutant into mice loss of ffunction (transgenic mice) How to create transgenic mice Normal Gene X Pronucleus from sperm Pronucleus from egg Normal unfused Fertilized mouse egg

23 Random integration of transgene (X*) Inject mutated t dx (X*) Unfused Fertilized mouse egg after injection

24 Transfer injected eggs Fused nuclei into foster mother Foster mother (contains ti X*) (screen transgenics by PCR) (Transgenic mice) LOOK FOR DEFECTS

25 Post-transcriptional techniques to manipulate gene function By selectively destroying mrna

26 Manipulation puato at the RNA level e Antisense approach RNAi (RNA-interference) approach

27 ANTIsense RNA DNA: 5 CGATCCGTATCGTAAAT 3 3 GCTAGGCATAGCATTTA 5 (coding strand) (template strand) mrna: 5 3 CGAUCCGUAUCGUAAAU Antisense mrna: 3 GCUAGGCAUAGCAUUUA 5 Sense and antisense mrna are complementary and hybridize Sense mrna: antisense mrna: CGAUCCGUAUCGUAAAU GCUAGGCAUAGCAUUUA dsrna (target for RNAse Mediated )

28 Practical approach Microinject or transfect chemically synthesized antisense RNA (finds the complementary mrna and leads to destruction of the target mrna) Transfect plasmid DNA that codes for antisense RNA Chromosomal DNA Plasmid DNA Sense mrna hybridize Antisense mrna destruction long double stranded RNA can lead to antiviral response

29 RNA interference (RNAi) A double stranded RNA leads to silencing of expression of target genes that are homologous to the dsrna was first observed in C. Elegans. drna dsrna dicer (ds RNA-dependent endonuclease) SiRNA (small interfering RNA) nt (19 nt+2 nt overhang on each end) Activated RISC (RNA-induced silencing Complex; recognizes sirna) Target mrna dicer only single strand of Si RNA Is held by RISC (ss RNA serves as a guide) dicer No translation (degradation of target mrna)

30 application of SiRNA High throughput genetic screening in disease models