NIH Filariasis Research Reagent Resource Center (FR3)

Experimental Techniques in Filariasis Research

 

09 - 13 March 2009

University of Georgia, College of Veterinary Medicine

 

Class Schedule

NOTE: All lectures and labs will be held in the VetLab (in back of reading room)

 

 

Instructors:

Ray Kaplan, DVM, PhD                    University of Georgia

Steve Williams, PhD                           Smith College

Shelly Michalski, PhD                        University of Wisconsin-Oshkosh

Andy Moorhead, DVM, PhD             University of Georgia

Mike Dzimianski, DVM, MS             University of Georgia

Prasit Supakorndej, PhD                     University of Georgia

Sue Haynes, MS                                 Smith College

John McCall, PhD                              University of Georgia

Steve Harvey, DVM, MS                   University of Georgia

Leanne Alworth, DVM, MS               University of Georgia

 

 

Support Personnel:

Bob Storey, MS, RVT and LaDonna Allen

 

 

Day              Time                  Event

 

Monday       8:30-9:00            Welcome and Introductions

 

                                 9:00-9:15            Lecture (Moorhead): FR3 parasite resources available

 

                     9:15-10:00          Lecture (Williams): FR3 molecular resources available: how to access these; web site, Use of reagents from the FR3 (libraries, clones, etc.).

 

                                 10:00-10:45        Lab (Dzimianski/Supakorndej): Use blood from cat with B. malayi microfilariae (mf) to set up membrane-feeders and start infecting mosquitoes.

 

                                 10:45-11:00        Break (Refreshments)

                                

                                 11:00-11:45        Lecture (Williams): The global effort to eliminate lymphatic filariasis

 

                                 11:45-12:30        Lecture (Michalski): Overview of nematode biology and introduction to filarioid nematodes

 

                                

 

                    

 

                                

 

                                 12:30-2:00          Lunch

 

                                 2:00-3:00            Lecture (Williams): Introduction to the isolation of DNA and RNA from filarial parasites. Special considerations compared to isolating DNA from other organisms.

 

                     3:00-3:45            Lab (Dzimianski/Supakorndej): Collect L3 from mosquitoes, examine L3 under microscope, inject L3 into jirds

 

                     3:45-4:00            Break (Refreshments)

 

         4:00-5:30            Lab (Williams/Haynes): DNA extraction

 

 

 

Tuesday      8:30-9:30            Lecture (Harvey/Alworth): Using gerbils in biomedical research

 

                                         9:30-10:30          Lab (Harvey/Alworth): Using gerbils in biomedical research

 

                     10:30-10:45        Break (Refreshments)

 

                     10:45-11:30        Lab (Williams/Haynes): Finish DNA extraction

 

                     11:30-12:30        Lecture (McCall): B. malayi / B. pahangi mosquito vector phase

 

                     12:30-2:00          Lunch

 

                     2:00-3:00            Lecture (McCall): B. malayi / B. pahangi vertebrate host(s) phase

 

                     3:00-4:30            Lab (Dzimianski/Supakorndej): Necropsy B. malayi infected jirds; Collect adult worms, L4 and microfilariae from peritoneal cavity and examine under microscopy. Identify and sort male and female worms.

 

                     4:30-6:00            Lab (Williams/Haynes): Isolate RNA from L3 worms.

                    

 

 

Wednesday 8:30-9:30            Lab: Pick mosquito pupae; put mosquito eggs in water for hatching into larvae.

 

                     9:30-10:15          Lab (Williams/Haynes): Finish RNA isolation from L3 worms.

 

                     10:15-10:30        Break (Refreshments)

 

                     10:30-12:00        Lecture (Williams): Details of gene expression analysis and RT-PCR, Analysis of EST and other types of expression data, Microarrays

 

                     12:00-1:30          Lunch

 

 

                     1:30-2:00            Lecture (Michalski): Transcriptome analysis I - applications (genome annotation, stage-specific expression, tissue-specific expression, pathogenesis)                                                                  

 

                     2:00-2:45            Lab: [check RNA on Nano-Drop spec]. Set up RT-PCR reactions.

 

                     2:45-3:15            Lecture (Michalski): Transcriptome analysis II - Methods (sequencing-,  hybridization-, and PCR-based, and Verification (RT-PCR, qPCR, in situ hybridization, immunohistochemistry)

 

                     3:15-3:30            Break (Refreshments)

 

                     3:30-4:15            Lecture (Michalski): Statistical Analysis (the quagmire of statistics)

and Reporting Results (who curates databases, who curates genomes, what is reportable?)

 

                     4:15-6:15            Lab (Michalski): Dissect freshly fed (2 hours prior) mosquitoes and quantify midgut penetration of L1. Dissect day 5.5 and day 9.5 mosquitoes – recover and examine L2 and L3 stages

 

                     7:00-                   Dinner at East-West Bistro

 

 

 

Thursday    8:30-10:00          Lecture (McCall): D. immitis mosquito vector phase, D. immitis vertebrate host(s) phase.

 

                     10:00-10:15        Break (Refreshments)

 

                     10:15-11:00        Lecture (McCall): Wolbachia

 

                     11:00-11:30        Lab (Dzimianski/Supakorndej): Set up mosquito larval cultures.

 

                     11:30-12:30        Lab (Williams/Haynes): Set up DNA PCR

 

                     12:30-2:00          Lunch

 

                     2:00-3:30            Lab (Dzimianski/Supakorndej): Necropsy of Brugia-infected jirds with lymphatic infection.

 

                     3:30-4:30            Lab: Set up and run gel on RT-PCR and DNA PCR and a second gel for filarial DNA and RNA.

 

                     4:30-4:45            Break (Refreshments)

 

                     4:45-5:45            Lab: Finish running gel on RT-PCR and DNA PCR. Stain and photograph gel, interpret data.

 

                     5:45-6:30           Lecture (Williams): Interpreting RT-PCR data. Q-RT-PCR.

 

 

 

 

Friday          8:30-9:15            Lecture (Williams): Finding clones in cDNA and genomic libraries. Bioinformatics.

 

                     9:15-10:15          Lecture (Williams): Bioinformatics of filarial data sets. ESTs and BLAST. Accessing the genomic data at TIGR. Genomics.

 

                     10:15-10:30        Break (Refreshments)

        

         10:30-12:00        Lecture (Williams): Using online bioinformatics resources. Finishing up unfinished business. END OF COURSE