presentation.pdf

A 10 minute presentation given during the Intermediate Hosts section of the 15th International Symposium on Schistosomiasis

Transcript

1. Nicolas J Wheeler, Nathalie Dinguirard, Theresa Maier; Erica KO Namigai, Josh Tycko, Jutta Reinhard-Rupp, Timothy P Yoshino, Mostafa Zamanian Progress toward transgenesis in Biomphalaria glabrata
2. !2 B. glabrata - host of Schistosoma mansoni Biomphalaria glabrata embryonic cell line
3. !3 History of genetic manipulation of B. glabrata
4. !4 Why transgenesis now? Driving endonuclease gene Endonuclease target site Cut Repair Population suppression or population replacement
5. !5 Goals 1. Develop transgenesis at the adult stage 2. Develop transgenesis at the egg stage 3. Re-establish and update the transgenesis protocol in the Yoshino lab strain of Bge
6. !6 1. Develop transgenesis at the adult stage ‣ Establish delivery techniques ‣ Injection of ovotestis ‣ Dye stains site of injection ‣ Rapid injection can cause dye to spread throughout the organism ‣ Injection does not reduce fecundity or cause mortality Clutches per Snail Eggs per Snail Survival 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 0 1 2 3 4 0 10 20 30 40 0.00 0.25 0.50 0.75 1.00 DPI Injected Uninjected p = 0.54 A B C D E. H. Michelson, Trans. Am. Microsc. Soc., vol. 77, no. 3, pp. 316–319, 1958.
7. !7 2. Develop transgenesis at the egg stage ‣ Establish delivery techniques ‣ Injection of multiple eggs per clutch ‣ Dye stains embryo and is retained in the egg ‣ Injection does not harm development or hatching
8. !8 3. Re-establish and update the transgenesis protocol in the Yoshino lab strain of Bge 94.9% 0e+00 1e+05 2e+05 0e+00 1e+05 2e+05 FSC.A SSC.A 96% 0e+00 1e+05 2e+05 0e+00 1e+05 2e+05 FSC.A SSC.A 4.84% 0e+00 1e+05 2e+05 0e+00 1e+05 2e+05 LiveDead eF780.A SSC.A 87.9% 0e+00 1e+05 2e+05 0e+00 1e+05 2e+05 FSC.A SSC.A 92.8% 0e+00 1e+05 2e+05 0e+00 1e+05 2e+05 FSC.A SSC.A 98.4% 0e+00 1e+05 2e+05 0e+00 1e+05 2e+05 LiveDead eF780.A SSC.A 0 3 6 9 Reagent Alone RNA Alone Untreated 2 4 6 8 10 12 14 uL jetPEI per 1 ug mRNA Percent Fluorescent DIC Hoechst 33342 | GFP A B C D ‣ jetPEI at higher ratios than previously used ‣ GFP-encoding mRNA ‣ 6-12% transfection efficiency ‣ Little to no cytotoxicity (though evidence of some cell lysis)
9. !9 Future Directions ‣ Injection snails ovotestis and eggs with higher ratios of transfection reagent, as optimized with Bge ‣ Inject snail ovotestis and eggs with lentiviral particles ‣ Develop sperm-mediated gene transfer using artificial insemination ‣ Transfect Bge with Cas9 mRNA and selected small guide RNAs to perform genome editing
10. Zamanian Lab Mostafa Zamanian Thank You Yoshino Lab Tim Yoshino Nathalie Dinguirard University of Wisconsin-Madison Erica Namigai Theresa Meier Josh Tycko Gabrielle Disselhoff Jutta Reinhard-Rupp Merck KGaA