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Tbx5 Dominant Negative Protein Inhibiting Myocardial Regeneration in Zebrafish Joshua Fung BNFO300 12/15/17.

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Presentation on theme: "Tbx5 Dominant Negative Protein Inhibiting Myocardial Regeneration in Zebrafish Joshua Fung BNFO300 12/15/17."— Presentation transcript:

1 Tbx5 Dominant Negative Protein Inhibiting Myocardial Regeneration in Zebrafish
Joshua Fung BNFO300 12/15/17

2 Introduction 610,000 people die of cardiovascular disease per year
Possible solution: Heart Regeneration Zebrafish: one of few organisms that possess this ability Tbx5: expressed in heart development

3 Dominant Negative Mutation
mutation that alters protein so it interferes with function of wild-type protein produced by other allele

4 Methods Zebrafish Purchase Tbx5 Dominant Negative construction
Heterozygous cmlc2-creEr(T2) transgene zebrafish Wild-type zebrafish embryos for tbx5DN transgene Tbx5 Dominant Negative construction CreLox Recombination

5 Methods Er(T2) Cre Hsp90 Cmlc2 (promoter, regulatory DNA sequences)
restricts the Cre only to the heart cmlc2 CreEr(T2)

6 Methods Zebrafish Purchase Tbx5 Dominant Negative construction
CreLox Recombination

7 Method β-actin STOP BFP (blue) mCherry (red) Tbx5 DN loxP loxP
Transgenic line: Tg(bactin2:loxP-mTagBFP-STOP-loxP-mCherry-tbx5dn)

8 Methods

9 Methods Zebrafish Purchase Tbx5 Dominant Negative construction
CreLox Recombination

10 Cre Er(T2) Hsp90 Cmlc2 (promoter, regulatory DNA sequences) restricts the Cre only to the heart cmlc2 CreEr(T2) 4HT Hsp90 Knocked off by 4HT hormone (4-hydroxytamoxifen) Hormone that allows CreEr to go to nucleus NUCLEUS β-actin STOP BFP (blue) mCherry (red) Tbx5 DN loxP loxP Transgenic line: Tg(bactin2:loxP-mTagBFP-STOP-loxP-mCherry-tbx5dn)

11

12 Methods continued Ventricular resection surgery- surgical injury
Allow for regeneration or scarring Euthanization- immersion in tricaine Perfusion- phosphate buffered formalin Cryosectioning- slide preparation Acid Fuchsin-Orange G Staining

13 Methods continued Ventricular resection surgery- surgical injury
Allow for regeneration or scarring Euthanization- immersion in tricaine Perfusion- phosphate buffered formalin Cryosectioning- slide preparation Acid Fuchsin-Orange G Staining

14 Methods continued Ventricular resection surgery- surgical injury
Allow for regeneration or scarring Euthanization- immersion in tricaine Perfusion- phosphate buffered formalin Cryosectioning- slide preparation Acid Fuchsin-Orange G Staining

15 Methods continued Ventricular resection surgery- surgical injury
Allow for regeneration or scarring Euthanization- immersion in tricaine Perfusion- phosphate buffered formalin Cryosectioning- slide preparation Acid Fuchsin-Orange G Staining

16 Methods continued Ventricular resection surgery- surgical injury
Allow for regeneration or scarring Euthanization- immersion in tricaine Perfusion- phosphate buffered formalin Cryosectioning- slide preparation Acid Fuchsin-Orange G Staining

17 Methods continued Ventricular resection surgery- surgical injury
Allow for regeneration or scarring Euthanization- immersion in tricaine Perfusion- phosphate buffered formalin Cryosectioning- slide preparation Acid Fuchsin-Orange G Staining

18 Results One of two things can happen:
Resected hearts scar or regenerate 1. Scarring indicates tbx5DN inhibits regeneration and that tbx5 is necessary (Bottom) 2. Successful regeneration indicates that tbx5 is not necessary for heart regeneration (Top) Figure5. Gupta et al, 2013

19 Synthesis and recap Expressing tbx5DN vs conditional knockout
If conditional knockout approach was used, which would be the “real” results

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