Source: LOUISIANA STATE UNIVERSITY submitted to NRP
THE APPLICATION OF RNAI KNOCKDOWN TECHNOLOGY TO FISH SYSTEMS USING THE INTERFERON INDUCED MX GENE AS A MODEL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0202340
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2004
Project End Date
Sep 30, 2005
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
PATHOBIOLOGICAL SCIENCES
Non Technical Summary
ALthough not highly prevalent CCV can cause massive losses to the catfish farming industry. To determine if catfish Mx has a role in immunity to CCV.
Animal Health Component
(N/A)
Research Effort Categories
Basic
70%
Applied
(N/A)
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113710109050%
3113710110150%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1090 - Immunology; 1101 - Virology;
Goals / Objectives
To determine if the channel catfish Mx gene plays an antiviral role against channel catfish virus (CCV).
Project Methods
Initially the Mx specific oligos will be synthesised and cloned into a vector able to synthesise short hairpin RNA's (shRNA) from the gene specific DNA fragments. A stably transfected channel catfish ovary (CCO)cell line will be created using shRNAMx. Control CCO cells will be transfected with a control plasmid containing a scrambled oligo sequence to create a permanent control cell line. Control CCO cells should produce large amounts of Mx when stimulated with the interferon inducer poly I:C, while the cells transfected with shRNAMx should not. The exact dose of poly I:C that will stimulate maximum Mx levels in the control cells will be determined and quantitative RT-PCR carried out. To determine that the knockdown cell line is truly knocked down the same quantitative RT-PCR will be used to measure Mx levels in response to poly I:C compared to control CCO cells. Following this a protection assay will be carried out on the Mx knockdown cell line. Cells will be stimulated with poly I:C followed by infection with CCV. It is likely that various doses of CCV will need to be tested within the dose of poly I:C chosen. This will determine if Mx is involved in immunity to CCV.

Progress 10/01/04 to 09/30/05

Outputs
To date 14 short hairpin RNA's (shRNA) have been designed, using online tools, to inhibit the function of the channel catfish Mx gene. The Mx gene is approximately 1900 nucleotides long and the shRNA's were chosen from various locations throughout the gene. The entire Mx gene was cloned into a plasmid encoding a reporter gene (alkaline phosphatase) and used to screen the shRNA's for effectiveness. Recently one shRNA was identified that effectively knocked down Mx gene function and work is ongoing in characterizing it. In preparation for an effective shRNA and the need to produce a stably transfected cell line expressing the Mx shRNA, transfection of channel catfish ovary (CCO) cells has been optimised. Transfection efficiency has been increased from less than 1% to approximately 40% and monitored by GFP expression. This level of transfection is high enough to enable the use of flow cytometry to select for cells expressing GFP to create the stably transfected cell line.

Impacts
The use of shRNA's could be very important in determining gene function in any organism. In the case of channel catfish, the most extensively farmed fish in the US, disease causes major economic losses. If we can understand the role individual catfish genes play in response to infection with certain pathogens it may be possible to limit these losses in the future with more effective treatments.

Publications

  • No publications reported this period