3.2 Microbiology/Parasitology
3.2.1 PBV 251
Programme Title: |
Transgenic worms for the control of possums. |
| Programme Leader: | Dr Chuck Shoemaker |
| Institution: | AgResearch, Wallaceville |
Programme Goal: To develop the technology to enable the possum parasite Parastrongyloides trichosuri (Pt) to be used as a vector for genes expressing proteins which could interfere with reproductive success, growth or longevity of it's host.
Objective 1
Objective Title: Cloning Pt promoters for use in expression vectors.
Research Leader: Dr Chuck Shoemaker
Description:
The researchers will complete work to construct expression vectors for Pt. They will add to the vectors that use Caenorhabditis elegans promoters by constructing two vectors utilising Pt promoters to drive expression of green fluorescent protein (GFP). The researchers already have genomic clones for several Pt genes and need next to identify the promoter regions and introduce them into the expression vector. They anticipate using one collagen gene promoter and one gut specific protease promoter, each normally driving expression of secreted gene products. They may choose the hsp70 Pt promoter in addition or to replace one secretory promoter. The new constructs will be tested in C. elegans by co-transfection with a rol-6 expression vector and testing worms having a roller phenotype for expression of GFP at different stages in their life cycle.
Objective 2
Objective Title: Microinjection transformation of Pt.
Research Leader: Dr Chuck Shoemaker
Description:
The researchers will continue their efforts to obtain introduced marker gene expression in Pt by microinjection of expression vector DNA into worms. They will use existing expression vectors using C. elegans promoters, and add to their pools of expression vectors by using Pt promoters as they become available in Objective 1. Efforts will include research into culture and manipulation methods which minimise trauma to the worms and maximise viable recovery. In addition, they will vary the maturity of female worms injected, the precise position of the injection and timing of male mating.
Objective 3
Objective Title: Pantropic retroviral transformation of Pt.
Research Leader: Dr Chuck Shoemaker
Description:
Pantropic infection of germ cells will result in integration of the expression vector DNA into the genome to generate stable transgenic worms. The researchers will continue their efforts to develop a method that permits stable, marker gene expression in Pt by microinjection of pseudotyped retrovirus (pantropic) vectors into the C. elegans nematode model. They will attempt different methods of introducing existing pantropic virus preparations at different sites in worms of different stages, especially concentrating on injection of eggs at the single to four cell stages. Successful generation of stable transgenics will be determined by PCR of progeny worm DNA to detect the presence of vector DNA.
Objective 4
Objective Title: Immunogenic protein characterisation.
Research Leader: Dr David Heath
Description:
A P. trichosuri specific 70-80Kd secreted protein produced by infective larvae during penetration of the host has been demonstrated to be immunogenic in infected possums. The goal of this objective is to fully characterise this protein using molecular biological methods. The work forms part of the PhD programme being undertaken by Jan Newton-Howes.
3.2.2 PBC 208
Programme Title: |
Riboflavin Carrier Protein (RCP): Identification, purification and biological consequences. |
| Programme Leader: | Dr Ken McNatty |
| Institution: | AgResearch, Wallaceville |
Programme Goal: To control the reproduction of possums by targeting the RCP.
Objective 1
Objective Title: Control of reproduction by targeting the riboflavin (vitamin B2) carrier protein (RCP).
Description:
RCP is a potential target for the immunosterilisation of possums. RCP binds to riboflavin (vitamin B2) which is required for the production of essential co-enzymes which are involved in many metabolic pathways. RCP enables riboflavin to cross cellular barriers and is essential for delivering riboflavin to the embryo. Immunisation of female rats, mice and monkeys against chicken RCP causes early embryonic loss at about the time of implantation. However, immunisation against RCP does not adversely affect the mother's health. RCP is also found on the acrosomal surface of sperm and is thought to be essential for delivering riboflavin to sperm. Immunisation of male rats and monkeys against chicken RCP causes male infertility. RCP is found in milk and may be involved in the transfer of riboflavin from the mother's blood to her milk. This may mean that immunoneutralising RCP could prevent the survival of a new-born pouch young possum by depriving it of riboflavin. RCP lends itself to several different delivery techniques including insertion into a parasite and incorporation of a fragment of the RCP gene into recombinant "virus-like particles" produced in plants.
RCP work covered in this programme has the following goals:
- Establishing that immunisation of female possums with chicken RCP results in reduced fertility or increased pouch young mortality.
- Obtaining purified possum RCP and a partial amino acid sequence of possum RCP.
- Cloning the cDNA of possum RCP and sequencing the possum RCP gene.
- Developing an assay which can measure plasma levels of RCP.
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