Bio-ARROW - SmartForm - Research Description
This section is for describing your research from a biosafety
perspective. It is not appropriate to copy and paste the research
description from a grant proposal, animal protocol, or other document
because we need different information.
Remember to enter
information about the work that is done outside of your main laboratory
(i.e. core laboratories). Describe what materials are used in the core
facility and who performs the procedures (i.e. your staff, core staff,
For protocols with multiple projects, please number each project in the description. Add the biosafety level, location if applicable, relevant biosafety concerns including the risk to people, details on recombinant activities and factors important in risk assessment for each project.If flow cytometry is used, please describe what kind of cells you plan to analyze or sort including viability (alive or fixed) and how the cells are manipulated (how they are generated, transfected, transduced, labeled, etc.).
Here is an example of a well written research description:
Coronavirus (CV) infections frequently cause disease. Our goal is to define aspects of virus-induced inflammatory responses that contribute to disease.
- We have created and continue to modify PCR-based methods for the identification various corona viruses from patients. To that end, we grow a variety of respiratory viruses to use as standards for developing diagnostic tests. We use commercial cloning vector pGEM-T Easy (Promega) for TA cloning of PCR products for molecular typing of clinical isolates. We will use the same methods for CV-BB strains.
- pGEX, pGEM and pET-based plasmids contain human coronavirus genes. Protein expression is under the control of the inducible tac promoter or T7lac and hybrid CMVie enhancer fused to the chicken β-actin promoter (pTriEx). These plasmids are used for expression of viral proteins in E. coli. They have the ampicillin-resistance gene for growing and selection in E.coli. Viral proteins are necessary for studying host-shutoff mechanisms of virus in eukaryotic cells and production of virus-specific antibodies.
- We are going to validate the CV receptor candidate gene(s) identified by overexpression in non-permissive cells by down-regulation of RNA expression (RNA interference). Since the differentiated human epithelial cells susceptible to viral infection cannot be transfected efficiently by lipofection or electroporation, we would like to transduce them with lentivirus vectors (HIV-1) encoding short-hairpin (sh) RNAs targeting candidate gene(s). We are planning to use commercially available third-generation Lentivirus vectors that are replication defective due to segregation of vector and packaging cassettes in several (at least 3-4) plasmids and deletion of viral accessory genes to minimize the risk of production of replication-competent lentivirus. The lentivirus will be produced in small quantities (laboratory scale) and applied only to cultured airway cells in vitro. (See attached appendix for figure) This will be done at BSL2 containment.
- Flow Cytometry—We are interested in the identification of coronavirus susceptible cell type in human cells. We will transfect cell cultures with an in vitro-transcribed RNA product that encodes for a full-length CV genome containing a fluorescent protein (e.g. Green or Enhanced Blue Fluorescent Protein (GFP, RFP-A respectively)) gene insert. Individual vectors are replication competent, but replicate at about one log lower efficiency than parent strains, and only infect about 1% of cells in a given Cell-1 culture. Fluorescence-activated cell sorting and return of cells will be used to sort GFP- or RFP-A -positive cells from the total cell population. Afterward, total RNA will be extracted from sorted cells for determination of cell type-specific transcripts via qRT-PCR. All work performed with cells before and after sorting will employ BSL2 safety precautions.