3.2 Microbiology/Parasitology
| Programme Title: | Transgenic worms for the control of possums |
| Programme Leader: | Dr Allen Gruenberg |
| Institution: | AgResearch, Wallaceville |
| Programme Goal: | To develop the technology to enable the possum parasite Parastrongyloides trichosuri to be used as a vector for genes expressing proteins which could interfere with reproductive success, growth or longevity of its host. |
Objective 1
| Objective Title: | P. trichosuri-specific gene promoters. |
| Research Leader: | Dr Allen Gruenberg |
Description:
Researchers will continue their studies towards obtaining P. trichosuri specific transcriptional promoters. They will search the databases for Caenorhabditis elegans collagen genes abundantly expressed at or close to the worm surface. Candidate sequences containing upstream promoter regions for these genes will be amplified by the polymerase chain reaction (PCR) from C. elegans genomic DNA and incorporated into constructs along with the green fluorescent protein reporter gene. Promoter activity will be assessed by transformation of C. elegans by micro-injection of these constructs. The screening of the P. trichosuri genomic library will continue. Researchers will screen for the analogues of both the C. elegans collagen genes described above and for other appropriate and characterised nematode genes. Once characterised, the putative promoter regions associated with these genes will be tested in P. trichosuri by micro-injection with constructs containing those sequences upstream of the reporter gene.
Objective 2
| 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. Characterising the sequence or this protein provides an alternative approach to obtain a potentially useful promoter for use in expressing gene products in a P. trichosuri vector. The existing P. trichosuri genomic library will be screened with an oligonucleotide probe obtained from the amino acid sequence of the characterised protein, and the upstream region containing the putative gene promoter will be identified and tested as described in Objective 1. This work forms part of a PhD research programme being undertaken by Jan Newton-Howes.
Objective 3
| Objective Title: | Pantropic Retroviral Vectors. |
| Research Leader: | Dr Allen Gruenberg |
Description:
Researchers will continue to experiment with the use of pseudotyped pantropic retrovirus vectors to establish integrated genetic transformation in C. elegans and if successful apply the technology to the transformation of P. trichosuri. Although researchers have routinely achieved transformation of C. elegans by microinjection of sequences within plasmid constructs, inheritance of the transformed sequences is always by way of extrachromosomal arrays and thus non-Mendelian. Clearly, this would not be of long-term value in P. trichosuri if it is to be ultimately used as a vector in possum biological control.
Over the previous year researchers have relied on a limited supply of pantropic vectors obtained from Dr Jane Burns of the University of California, San Diego. This year, they expect to have high titred stocks produced in their own laboratory available that will allow greater progress. Focus will be placed on microinjection efforts on in utero fertilized eggs rather than the gonad syncytium, in the belief that active binding to intact cellular membranes by the virus is a requirement for introduction of transforming DNA sequences into the nuclei of germ cells.
| Programme Title: | Riboflavin Carrier Protein (RCP): Identification, purification and biological consequences. |
| Programme Leader: | Dr Ken McNatty |
| Institution: | AgResearch, Wallaceville |
| Programme Goal: | To induce fetal wastage and/or neonatal mortality in possums by blocking the transfer of maternal riboflavin to the fetus and/or newborn. |
Objective 1
Description:
This objective will:
- Attempt to isolate bovine RCP and partially sequence; develop assay for measuring possum RCP.
- If bovine RCP purification has been successful attempt purification of possum RCP for immunocytochemical and physiochemical analyses; characterise putative possum RCP activity in plasma and milk from possums in various reproductive states.
- If bovine RCP is not successful - continue further purification steps if options remain open. If not attempt a molecular biology approach.
- Possum RCP cDNA evaluation following RT-PCR and RNA purification from possum livers and/or reproductive tract.
Contact for Enquiries
Farm Monitoring Programme Manager
Monitoring and Evaluation
MAF Policy
PO Box 2526
Wellington
NEW ZEALAND
Phone: +64 4 894 0623
Fax: +64 4 894 0741
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