Neutrophil-specific Overexpression of FCHO2, a PCH family protein, in Danio rerio Chelsey Warning and Kate Cooper, PhD Loras College Department of Biology Fluorescence of zebrafish embryos following injection After multiple rounds of zebrafish embryo injections, overexpression of FCHO2 in neutrophils was not seen (Figure 4). DISCUSSION Site-directed mutagenesis of FCHO2 was successful FCHO2 was successfully cloned into a neutrophil-specific vector. Ligation and transformation successfully performed. Injections although have not led to overexpression of FCHO2 in neutrophils thus far, further research can be done using the newly synthesized vector in the future in hopes of learning the role of FCHO2 in cell migration. A standard miniprep was performed in order to extract desired DNA. In order to analyze and test the miniprep, a double restriction enzyme digest was performed using KpnI and BamHI. These digests were then run through gel electrophoresis on 0.8% agarose gels. Mating and Injection of Danio rerio After mating Danio rerio, the fertilized eggs were collected and washed with 1xE3 solution. Capillary tubes were modified into needles and loaded with LysC-FCHO2 DNA. It was then placed in the General Valve Corporation Picospritzer II microinjector. The embryos were placed in an injection ramp and the microinjector was then used to inject each embryo with 500 picoliters of the diluted DNA. RESULTS Successful Mutagenesis and PCR of FCHO2 An extra BamH1 site located in the middle of the FCHO2 gene was removed in order to perform restrictive enzyme digest in the future. PCR (polymerase chain reaction) was performed and gel electrophoresis was run to confirm success. The band located around 4500 bp is what was expected and was excised for future manipulation (Figure 1). KpnI and BamHI Restrictive Enzyme Digest After mutating the unwanted BamHI site within the FCHO2 gene the LIC-GFP-FCHO2 vector and the LysC vector was cut with BamHI and KpnI during two separate restrictive enzyme digests and run through gel electrophoresis. After the second digest with KpnI bands were expected at 3,500bp for the FCHO2 and about 6,000bp for the vector digest (Figure 2). Test Digest of Synthesized FCHO2 Vector A double restrictive enzyme digest was used on the plasmid mini-prep DNA using KpnI and BamHI, and the samples were run through gel electrophoresis. Bands were expected around 3,000bp and 5,000bp (Figure 3). INTRODUCTION Although cell migration is crucial to many cell functions, there is still a great deal of mystery surrounding the mechanics of this process. The Pombe cdc 15 homology (PCH) family of proteins have been shown to be involved in cell migration, but the exact role of these proteins is unknown. One such PCH family member has been shown to regulate the motility of neutrophil-like cells (Cooper, 2008). Although PCH family proteins are not closely related in terms of sequence, all members of this PCH family of proteins contain some similar coding regions, like the F- BAR domain (Heath, 2008). Some of the PCH family proteins have been found to play a role in these cellular processes (Chitu and Stanley, 2007), but there is still much we do not understand about this family of proteins. This project is focused on FCHO2 and the possible involvement it has in neutrophil cell migration through overexpression in Danio rerio. METHODS n Mutagenesis of BAMHI site within FCHO2 gene Polymerase Chain Reaction (PCR): For this protocol, new primers were originally designed and then made by Integrated DNA Technologies. A 50ng sample was prepared using 2 μl of 25ng/μl LIC-GFP-FCHO2, and a 100ng sample was prepared using 4μl of 25ng/μl LIC GFP FCHO2. In order to complete the mutagenesis process, a QuickChange protocol was used along with the solutions provided by the kit. The samples were then placed in the thermocycler to produce a PCR product with the site removed. Dpn1 was used to digest the PCR product and destroy previously methylated DNA. The DNA was then transformed using 1.5 µl of the 50ng or 100ng and 3 µl of the 50 ng and 100ng sample on LB plates with Kanamyacin at 37º overnight. After this step, 6 colonies were selected and a standard miniprep was used to extract DNA. A restrictive enzyme digest with BamHI was then used to ensure the success of removing the unwanted cut site in the middle of FCHO2. The products of the digests were run on a 0.8% agarose gel. n Cloning of FCHO2 and into a suitable vector for overexpression in specifically neutrophil cells Polymerase Chain Reaction (PCR): For this process, 1 μl of the previously mutated and diluted Lic-GFP-FCHO2 was mixed with standard solutions for PCR. The mixture was then placed in a Thermocycler. The purified PCR- FCHO2 product and a neutrophil specific vector LysC was cut in two separate restriction enzyme digests using BamHI and KpnI and ligated using a standard procedure. After ligation, a standard transformation was performed with both the ligated material as well as a control mixture on ampicillin plates. Bacterial colonies were picked and grown in LB broth with a concentration of 50 μl/ml of Ampicillin at 37ºC overnight. LORAS.EDU WORKS CITED Chitu, V., Stanley, E.R..Pombe Cdc15 homology (PCH) proteins: coordinators of membrane–cytoskeletal interactions. Trends in Cell Biology-1 March 2007 (Vol. 17, Issue 3, pp ). Cooper, K. M., Bennin, D. A., & Huttenlocher, A. (2008). The PCH Family Member Proline-Serine-Threonine Phosphatase–interacting Protein 1 Targets to the Leukocyte Uropod and Regulates Directed Cell Migration. Molecular biology of the cell, 19(8), Heath, R. J., & Insall, R. H. (2008). Dictyostelium MEGAPs: F- BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles. Journal of cell science, 121(7), ACKNOWLEDGMENTS I would especially like to thank Dr. Kate Cooper for her guidance and help throughout my undergraduate research. Another special thank you to Dr. Speckhard for his help during the Intensive Research J-term. I would also like to thank all of the Science Hall Faculty and Staff for their help in making my research practices as successful as possible. Figure 1: PCR of FCHO2 after mutagenesis yields one consistent band around 5000 base pairs (bp), which is the expected size of the replicated gene. PCR was performed as described in methods section but with FCHO2. These bands are visualized with ethidium bromide/UV light. The bands seen around 5000bp in lanes 2,3, and 4 were excised for further testing. The DNA ladder can be seen in lane 1. Figure 2: PCR and LysC vector cut with KpnI after being cut with BamH1. This gel electrophoresis was performed after both restrictive enzyme digests of FCHO2 and LysC. The bands are visualized with ethidium bromide and UV light. Compared to the ladder (lane 1) the FCHO2 vector (lanes 2,3,4) the expected band length around 3,500bp is seen. The expected band length in the LysC vector (5,6,7)is also seen around 6,000bp. Extra band around 1000bp is extra DNA removed from LysC vector. Figure 3: Gel electrophoresis follow test digest with KpnI and BamHI of synthesized vector after ligation and transformation The correct miniprep plasmid (lane 7) exhibits the bands at 3,500bp and 5,500 bp pairs as expected when compared to the ladder (lane 1). This miniprep was then sequenced to be further analyzed. The bands were again visualized with ethidium bromide and UV light. PhaseGFP Injected Uninjected