For on campus investigators use Department and Mail Code. For off campus investigators provide complete mailing address.

Transcription

1 FORM A - Protocol for Use of Recombinant or Synthetic Nucleic Acid Molecules in Research Idaho State University, Office for Research Institutional Biosafety Committee (IBC) 65 Alvin Ricken Drive, Pocatello, ID Phone: Fax: Version.0 OFFICE FOR RESEARCH USE ONLY DATE RECEIVED OR REVISED: VERSION: *ONLY TYPED FORMS WILL BE ACCEPTED* If this protocol is a resubmission, which protocol(s) is it intended to replace: Old IBC Protocol No.(s): DATE APPLICATION COMPLETED: 2/20/203. PROJECT TITLE: Mechanisms of Insecticide Resistance 2. PRINCIPAL INVESTIGATOR: Name (Last, First): Campus OR Work Phone Number: Groome, James Department Affiliation: BIOLOGICAL SCIENCES Mailing Address : 650 Memorial Drive IBC No. (Will be assigned initially by IBC office): Choose One, if Applicable: New Address - Change for all other Address (use ISU if ISU employee or student): groojame@isu.edu Gale Life Science Bldg Idaho State University Pocatello, ID active protocols For on campus investigators use Department and Mail Code. For off campus investigators provide complete mailing address. 3. PERSONNEL for CORRESPONDENCE: List personnel below who should be copied on the correspondence. Name (Last, First): Name (Last, First): Name (Last, First): Groome, James Campus or Work Phone Number: Campus or Work Phone Number: Biological Sciences Department Affiliation: Department Affiliation: groojame@isu.edu Address (use ISU if ISU employee or student): Address (use ISU if ISU employee or student): 650 Memorial Drive Gale Life Science Bldg Idaho State University Pocatello, ID Choose One, if Applicable: New Address - Change for all protocols Mailing Address : Mailing Address : Choose One, if Applicable: New Address - Change for all protocols Choose One, if Applicable: New Address - Change for all protocols Research Coordinator (Not Directly Involved with rdna/ia) Research Coordinator (Not Directly Involved with rdna/ia) Research Coordinator (Not Directly Involved with rdna/ia) ALL personnel who work directly with recombinant or synthetic DNA must be listed in Personnel Section (VII). Page of 9 Document #

2 I. Review Category Based on the review categories defined in appendix X of this form, this protocol falls under: Review Category Review Category II Review Category III Review Category IV II. Background a. Purpose of the research project: Describe the overall purpose of the project in a few sentences. Use language and define scientific terminology in lay terms: The project involves the introduction of site-specific mutations into voltage gated sodium channel genes of insect neuronal, and mammalian neuronal and skeletal muscle tissues. The mutations will be those identified in the native, or wild type fruit fly (Drosophila) voltage gated sodium channel, that confer insecticide sensitivity to those channels, and which are not found at the homologous sites in the mammalian sodium channels. The purpose of the mutagenesis is to create mutations in the mammalian sodium channel that may confer sensitivity to insecticides, as will be tested functionally. Those mutations introduced into the mammalian sodium channel that significantly increase the sensitivity to the insecticide deltamethrin will be catalogued as potential single nucleotide polymorphisms (mutations) that would pose a health risk for in individuals that work with these compounds. This database will be useful in screening for insecticide sensitivity in individuals working with deltamethrin or related pyrethroid insecticides. b. Scientific background: Describe the scientific background and expertise of the personnel listed in this protocol: My laboratory has worked with standard microbiological procedures here at Idaho State University since These include the proper use of, containment, and disposal of agar plates and liquid cultures for growing E. coli competent cells for the purpose of propagation of DNA. My previous training in molecular biology and microbiology techniques including sterile technique is most heavily from a faculty position at Harvey Mudd College in Claremont, CA for which I was required to instruct students in the proper use of cells and media for cell biology and molecular biology laboratories. Training was received from Dr. Mary Williams at that institution with whom I was a coinstructor for Cell Biology and Genetics, and from Dr. Nancy Hamlett with whom I was a co-instructor for Introductory Biology. For each of these courses, both molecular and microbiological techniques including containment and disposal were a point of emphasis. I learned the technique of in vitro transcription to create messenger RNA for expression of protein in the laboratory of Dr. Peter Ruben at Utah State University in Logan, UT when I was a Research Assistant Professor there, and have used this technique since 990. Page 2 of 9 Document #

3 IIIa. Project Description - Table - Plasmids, Vectors, and Genes Antibiotic Plasmid/Source Resistance Gene pgemhe, Dr. Steven Cannon, University of Texas SW Medical Center, Dallas, TX pgemhe Dr. Steven Cannon, University of Texas SW Medical Center, Dallas, TX pgh9 Dr. Lori Isom, Department of Pharmacology, University of Michigan, Ann Arbor, MI pgh9 Dr. Ke Dong, Department of Entomology and Neuroscience, Michigan State University, East Lansing, MI pgh9 Dr. Ke Dong, Department of Entomology and Neuroscience, Michigan State University, East Lansing, MI Gene/Host/Source SCN4A gene ID SCN4A 25722; Rattus norveigicus, source same as plasmid SCN2A gene ID SCN2A 24766; Rattus norveigicus, source same as plasmid SCNB gene ID SCNB 29686; Rattus norveigicus, source same as plasmid para gene ID 3269; Drosophila melanogaster, source same as plasmid tipe gene ID 38504; Drosophila melanogaster, source same as plasmid Name/Function rat skeletal muscle voltage gated sodium channel, alpha subunit, electrical signaling rat brain type II voltage gated sodium channel, alpha subunit, electrical signaling rat brain voltage gated sodium channel, beta subunit, accessory protein fruit fly Drosophila neuronal voltage gated sodium channel, alpha subunit, electrical signaling fruit fly Drosophila neuronal voltage gated sodium channel, beta subunit, accessory protein Host Risk Group IIIa. Project Description - Table 2 - Bacterial Strains Strain/Source Escherishia coli XL-Gold ultracompetent cells, Agilent Technologies, Cedar Creek, TX Relevant Characteristics, Attenuation, etc tet r (mrca)83 (mcrcb-hsdsmr-mrr)73 enda supe44 thi- reca gyra96 rela lac Hte. tetracylcine resistant; endonuclease deficient; recombination deficient. mrca mrccb and mrr mutations prevent cleavage of methylated DNA such that DpnI can be used to cleave parental DNA after amplifiication in mutagenesis. lac sequence permits blue white screening. Derivative of Stratagene E.coli ultracompetent cells XHL-Blue MRF Native Antibiotic Resistance Gene Risk Group tet Plasmids To Be Transfected pgemhe pgh9 Page 3 of 9 Document #

4 IIIa. Project Description - Table 3 Non-Bacterial Strains and Cell Lines Strain/Source Relevant Characteristics Native Antibiotic Resistance Gene Risk Group Plasmids To Be Transfected IIIa. Project Description - Table 4 - Viral Packaging and Attenuation Virus Class and Name Source Packaging Cell Line Host Range Post Packaging Replication Defective Relevant Characteristics/Attenuation Mechanism Page 4 of 9 Document #

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6 IIIb. Project Description - Provide a description of the work conducted with the reagents listed in tables -4 above The project entails the incorporation of mutant amino acid residues into the mammalian voltage gated sodium channels SCN4A and SCN2A, and the Drosophila voltage gated sodium channel para. The mutations chosen are to. Mutate the Drosophila voltage gated para sodium channel at residues for which mutations in local mosquito population genetic screening suggest insecticide resistance, in a separate study. 2. Mutate the mammalian channel at sites homologous to that in the Drosophila sodium channel for which insecticide sensitivity is confirmed and for which resistance is confirmed in local mosquito population. The protocol for incorporating mutations into the genes for Drosophila or mammalian voltage gated sodium channels is polymerase chain reaction based mutagenesis. Primers are designed that incorporate mutations at point loci for single codons. These are then used in polymerase chain reaction amplification of the native gene to produce multiple copies of the gene with mutation added at the specific site. Native template gene sequences are then removed with DpN I restriction enzyme. The PCR products are used to transform the XL-Gold ultracompetent E. coli cells. Ampicillin-laced agar plates are used to screen for successful transformants as evidenced by colonies that grow in the presence of the antibiotic, as the plasmid vectors contain the resistance gene. These colonies are then grown in LB medium with to propagate the DNA. DNA is then used to direct the synthesis of messenger RNA using in vitro transcription. Messenger RNA is injected into oocytes of the African clawed frog Xenopus laevis for expression of the native or mutant proteins, for analysis using electrophysiology. IV. Animal Work a. Transgenic/Knockin/Knockout Generation of Rodent and Nonrodent species- Provide a complete description of the construct and species that will be used for production of transgenic/knockin/knockout animals. Indicate whether PI or another source will produce these animals. b. If transgenic/knockin/knockout animals will be purchased, transferred, or existing lines will be crossed- Provide a description below. c. All other use of rdna/synthetic DNA in Animals- Page 6 of 9 Document #

7 V. Plant Work a. Transgenic Production- Provide a description of the construct that will be used for production of transgenic plants. b. All other use of rdna/synthetic DNA in Plants- VI. Additional Information a. Indicate the location of ALL lab(s)/rooms in which the work will take place: Room No. 32, Building Gale Life Science; Room No. 463, Building Gale Life Science b. If applicable, indicate in which animal facility animal work will take place and type of room in which animals will be maintained. Facility Name. BSL Standard Housing c. Indicate the location of ALL lab(s) in which the rdna molecules/infectious agents will be stored: Check here if same as VIa Sciences Room No. 32, Building Gale Life Sciences; Room No. 255, Building Gale Life d. Indicate the highest biosafety level (BSL) required for this project: 2 e. Will shipping off-campus of any potentially infectious biological material (PIBM) be done? Yes No Name of person(s) responsible for shipping: VIIa. Personnel - List the following information for all personnel involved with this protocol. # ) Full Name: Groome, James 2) groojame@isu.edu 3) Degree: PhD 4) BST Date: 5) BBP Date: 6) ST Date: 7) Procedures/Experience with Procedures: Procedures/Experience with Procedures: The procedures with recombinant DNA include site-directed PCR-based mutagenesis, transformation of competent cells, screening of clones, propagation of plasmid DNA, purification of DNA, linearization of DNA, and in vitro transcription of messenger RNA. I have been using site-directed mutagenesis in my research since 2000, learning the techniques at both Harvey Mudd College as part of senior thesis supervision and teaching laboratory preparation, and at Utah State University as a researcher in summer months. All of the above 8) Trainer: Drs Mary Wlliams and Nancy Hamlett, Harvey Mudd College, Claremont, CA Page 7 of 9 Document #

8 procedures have been routine elements of my laboratory research since summer 2004 at Idaho State University. #2 ) Full Name: 2) 3) Degree: 4) BST Date: 5) BBP Date: 6) ST Date: 7) Procedures/Experience with Procedures: 8) Trainer: #3 ) Full Name: 2) 3) Degree: 4) BST Date: 5) BBP Date: 6) ST Date: 7) Procedures/Experience with Procedures: 8) Trainer: #4 ) Full Name: 2) 3) Degree: 4) BST Date: 5) BBP Date: 6) ST Date: 7) Procedures/Experience with Procedures: 8) Trainer: #5 ) Full Name: 2) 3) Degree: 4) BST Date: 5) BBP Date: 6) ST Date: 7) 8) Trainer: Page 8 of 9 Document #

9 VIIb. Personnel Training Describe the training provided to your personnel. For protocols involving biosafety level 2, please describe all additional training. For this project all procedures are performed by the PI. VIII. Assurances The Principal Investigator assures that the use of all rdna, potentially infectious agents, and infectious agents will be conducted in accordance with the ISU Institutional Biosafety Committee Policy. Signature of Principal Investigator Date 2/20/203 Typed or printed PI name: James R. Groome Page 9 of 9 Document #