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 as different information is needed.
Here are some things to consider when writing your research description:
Describe what you are doing:
- · What microbes, cells, animals, plants, genes, plasmids, toxins, and systems are you using?
- · In what combination are the plasmids, genes, systems, cells manipulated?
- · What procedures and equipment are used?
Identify special considerations for a project or activity:
- · Do you have a specific space that you use for an experiment?
- · Is specific containment equipment used?
- · Will the activity require different PPE?
- · Will the activity require a specific piece of equipment?
- · Will activity require specific training?
- · Change in risk to person, environment, public, or animals?
- · Special hazard communication?
Identify the recombinant materials:
- · How are they put together? (genes, system, microbes)
- · What modifications are made and are there any expected changes in pathogenicity, virulence, host range, or antibiotic resistance caused by the modifications?
State collaborations with another lab or a core facility:
- · On or off campus?
- · What activities will your lab perform?
- · What activities will the collaborator/core perform?
- · What materials will you provide to the collaborator/core? (e.g., fixed, material type, modifications)
- · What modifications were performed on the materials prior to giving/sending them to the collaborator/core?
- · What modifications will the collaborator/core perform on the materials?
- · Will any materials be returned to you from the collaborator/core?
- · Are there any special hazard communication, disposal, spill clean-up, or other special considerations for the materials shared with collaborator/core?
Here are examples of well written projects within research descriptions:
Project 1: Bacterial cloning
E. coli K12 and BL21 will be utilized to propagate bacterial plasmids (pET-based). These plasmids will be used for expression of various proteins in E. coli. Plasmids have the ampicillin-resistance gene for growing and selection in E.coli. We prepare these plasmids through standard cloning methods, including transforming the bacteria, expanding the cultures and isolating plasmid DNA and/or proteins from the bacterial cultures. No toxin genes or oncogenes will be expressed.
Project 2: Natural antibiotic resistance
To understand the mechanism of pathogenicity, Staphylococcus aureus USA 300 and clinical isolates of S. aureus will be studied. DNA/RNA is extracted and then used for PCR, sequencing, and metagenomics. Genes of interest will also be identified using random mutant libraries consisting of transposons that confer chloramphenicol resistance.
S. aureus is a human pathogen and will be handled at BSL2. Containment will be used as described in the ‘Containment’ section. Since strains of S. aureus can have natural occurring drug resistance, clinical isolates will be tested for antibiotic resistance. The susceptibility profiles of the isolates are attached. S. aureus USA 300 is known to be methicillin resistant.
Project 3: Yeast
The molecular function of genes will be tested in Saccharomyces species. Several auxotrophic and drug-resistance markers will be introduced on plasmids or through stable integration of DNA into the genome. Conventional molecular biology approaches as well as CRISPR/Cas9 are used. rDNA techniques include genome-wide screens, gene overexpression, gene knock-down, as well as specific DNA deletions, additions, and point mutations. Precautions used for experiments that use gene drive are described in the ‘Recombinant Materials’ section.
Project 4: Viral Vectors
To understand the function of the oncogene PI3K, we will use CRISPR to modify the point mutation within PI3K to remove the mutation (thus restoring gene function). It is not anticipated that by removing the point mutation in PI3K that this would cause an increase in risk to people working with the cell line.
To do this we will use one of several viral vector systems (see construct section for details). The viral vectors we use are rendered defective by using multi-plasmid systems. The vector-systems used contain deletions in essential replication and structural genes, so that the resulting vectors support only one round of integration and are unable to further replicate.
We use the second generation HIV based viral vector (as listed in construct section) which is a three plasmid system. The tat and rev genes are on a separate plasmid from the gag and pol genes. Accessory genes vif, vpu, vpr and nef have been removed. 3’ LTR is present and replication competent virus generation would require several recombination events.
We also use a third generation HIV based lentiviral vector system as a four plasmid system. The rev gene is on a separate plasmid from gag and pol. Accessory genes vif, vpu, vpr and nef have been removed. The 3’ LTR-SIN has been deleted and replication competent virus generation would require four recombination events.
For both the second and third generation, the envelope gene is VSV-G as it enters a variety of cells. HEK293 is used as the packaging cell line. The second and third generation systems will not be mixed together to prevent the formation of replication competent viruses. Both systems will be used with cell lines listed in the ‘Cell Culture’ section with genes listed in the ‘Genes and DNA/RNA fragments’ section.
The moloney murine leukemia virus (MMuLV)-derived retroviral vector is also utilized and may be modified so that it is able to enter human cells through the VSV-G envelope gene. It is a two plasmid system and PT67 and HEK293 are packaging cell lines (providing gag and pol). This vector system will only be used with HEK293 cells with gfp and Akt or mTOR.
We express CRISPR guide RNAs and CAS from a single construct, and homology dependent repair cassettes from two separate plasmids. The sequences of guide RNAs and homology repair cassettes are specific for the species-origin of suitable host cells for any given virus under study. These include human-derived cells. The CRISPR system is a single vector system and does not contain homologous DNA surrounding either the Cas9 or the guide RNAs. No gene drive issues with this system as this is for cell culture only.
Since the PI3K is a known oncogene, persons working with the HIV based viral vector systems and CRISPR will be apprised of the possibility that the vector has the capability to enter a human cell. They will be provided information on this research in case they would like to follow up with a medical professional for questions or in the event of an exposure. (Specified in the ‘Emergency Response’ sections and ‘Occupational Health Considerations’ sections of this protocol)
Project 5: Cell Culture and Animals
Project 5A: In order to study genes involved with tumor progression in squamous cell carcinoma (SCC) in vitro we will utilize immortal mouse and human tumor tissue culture lines that we have generated (mSCC and hSCC respectively). To assess the relevance of genes of interest (GOI) as possible tumor promotor genes in these cells we will add siRNA to knock-down the GOI. Scrambled siRNA will be used as a control. Knocking down a tumor promotor gene should allow for decreased growth of the cells. Once we have genes that we feel are relevant we will confirm them in primary cells from fresh tumors. We will also study the mechanism in more detail by upregulate the selected tumor promotor as further described in 5B. This work will all be done at BSL2 in a BSC. Fixed samples will be transported to UWCCC Experimental Pathology Laboratory for histology. Once cells are lysed protein work can be performed on the bench.
Project 5B: Once we have selected genes that act as tumor promotors in our cell lines we will screen multiple established tumor cell lines from ATCC by using the siRNA as described in Project 5A. This project will have all the same safety precautions as Project 5A.
Project 5C: The in vitro studies will be followed by experimentation in vivo. We have established transgenic mouse lines that produces SCC tumors. We will initiate tumor growth and then at predetermined tumor size will give the siRNA or control scrambled siRNA via tail vein injection. Tumor harvesting will be performed according to predetermined criteria. This work will be done at ABSL1. Analysis will include imaging of animals, tumor histology, and Western blot. The UW-Madison Small Animal Imaging Facility (SAIF) core will be informed of the ABSL1 status. Animals will be transported according to our Transport protocol and will be housed in quarantine after the imaging sessions. Histology samples are fixed and therefore can be handled at BSL1 by UWCCC Experimental Pathology Laboratory personnel.
Project 5D: We will assess relevance of the selected gene for clinical situations using a Patient Derived Xenograph (PDX) model. Human tumors will be received from an IRB approved protocol. These will be implanted into the flanks of NSG mice to be propagated. According to pre-set criteria, the tumors will be harvested in a BSC. A small portion will be fixed and sent for histology, a small portion will be froze back, and the remainder will be passaged into the flanks of Nude mice. When passaged tumors reach experimental criteria the siRNA or scrambled siRNA will be given to the Nude mice by tail vein injection. Scheduled imaging will be performed on live mice at the UW-Madison Small Animal Imaging Facility (SAIF) at ABSL2. Animals will be transported according to our Transport protocol and housed in quarantine after imaging sessions. Tumors will be harvest in a BSC according to pre-set criteria and samples sent for fixed histology and used for protein analysis as described above. Sorting of live cells will be performed at the UWCCC Flow Cytometry core by core staff.
Project 6: Plants
Our research goal is to determine the genes and mechanisms responsible for the colonization of plants by Salmonella. Various Salmonella mutants will be generated using standard genetic techniques (chemical mutagens and transposable elements), including point mutations and gene deletions in putative colonization genes. Complementation studies may be performed using the plasmids listed in the ‘Constructs’ section. Salmonella mutants may be tetracycline and/or kanamycin resistant. No increase in virulence is anticipated. Plants will be inoculated in a BSC with 106 CFUs Salmonella (wild type or mutant) per plant at different stages of growth. Plants will be grown in plant growth chambers located in aBSL-2P laboratory, see attached picture, under various light cycles and temperatures. Samples of plant tissues will be handled in the BSC for harvesting, homogenization, and analysis by plating and bacterial enumeration at various stages post inoculation. All transgenic plants are destroyed by autoclaving prior to flowering.