Genera.on, cloning, and expression of full-­‐length human EVC gene Trey Polvadore, Ka Choi, and Odutayo Odunuga Department of Chemistry, Stephen F. AusBn State University Introduc.on: Results: •  Ellis van-­‐Creveld Syndrome (EvC) is a rare condiBon that is characterized by short limbs, polydactyly, nail dysplasia, and other developmental afflicBons.1 •  MutaBons in EVC and EVC2 genes lead to the development of EvC.1,2 •  EVC and EVC2 proteins are posiBve modulators of the Indian Hedgehog signaling pathway which regulates the growth of bones and related Bssue.3,4 •  The two proteins interact with each other and localize on the primary cilia membrane.4 •  The precise role of the proteins in the Indian Hedgehog signaling pathway has not yet been determined. complete EVC gene: Strategy to generate B A C D Future Work: •  Confirm DNA sequence of human EVC gene product Figure 1. GeneraBon and cloning of the full-­‐length hEVC gene from human heart cDNA library, including (A) overlapping fragments, (B) fragment sizes, (C) overlap extension PCR, and (D) ligaBon into vectors. •  OpBmize expression protocol •  Characterize biochemical properBes of hEVC protein 1000 bp è 750 bp è Figure 2. IsolaBon of Fragments 2 and 3, 787 bp and 777 bp respecBvely, from human heart cDNA library via PCR. A B 2500 bp è 1500 bp è 1000 bp è 750 bp è 1500 bp è 1000 bp è 750 bp è Figure 3. GeneraBon of full-­‐length hEVC gene from three fragments. (A) Fragments 2 and 3, 787 bp and 777 bp respecBvely, were ligated to form Fragment 4, with a length of 1467 bp, via SOE-­‐
PCR. (B) Fragments 1 and 4, 1537 bp and 1467 bp respecBvely, were ligated to form the full-­‐length hEVC gene, with a length of 2976 bp, via SOE-­‐PCR. A B 3000 bp è 1000 bp è C D Acknowledgements: Stephen F. AusBn State University •  Chemistry Department •  Office of Research and Sponsored Programs Figure 4. Cloning and expression of full-­‐length hEVC gene, involving (A) pGEMT-­‐Easy vector map, (B) confirmaBon of construct via double digesBon, (C) Bme scale expression analysis with SDS-­‐PAGE, and (D) p202 vector map. References: 1.  Ruiz-­‐Perez, V.L.; Tompson, S.W.; Blair, H.J.; Espinoza-­‐Valdez, C.; Lapunzina, P.; Silva, E.O.; Hamel, B.; Gibbs, J.L.; Young, I.D.; Wright, M.J.; Goodship, J.A. MutaBons in two nonhomologous genes in a head-­‐to-­‐head configuraBon cause Ellis-­‐van Creveld syndrome. Am J Hum Genet 2003, 72(3), 728-­‐732. 2.  Ruiz-­‐Perez, V.L.; Ide, S.E.; Strom, T.M.; Lorenz, B.; Wilson, D.; Woods, K.; King, L.; Francomano, C.; Freisinger, P.; Spranger, S.; Marino, B.; Dallapiccola, B.; Wright, M.; MeiBnger, T.; Polymeropoulos, M.H.; Goodship, J. MutaBons in a new gene in Ellis-­‐van Creveld syndrome and Weyers acrodental dysostosis. Nat Genet 2000, 24(3), 283-­‐286. 3.  Ruiz-­‐Perez, V.L.; Blair, H.J.; Rodriguez-­‐Andres, M.E.; Blanco, M.J.; Wilson, A.; Liu, Y.N.; Miles, C.; Peters, H.; Goodship, J.A. Evc is a posiBve mediator of Ihh-­‐ regulated bone growth that localises at the base of chondrocyte cilia. Development 2007, 134(16), 2903-­‐2912. 4.  Blair, H.J.; Tompson, S.; Liu, Y.; Campbell, J.; MacArthur, K.; PonBng, C.P.; Ruiz-­‐Perez, V.L.; Goodship, J. Evc2 is a posiBve modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus. BMC Biology 2011, 9(14)