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Abstract Detail


Plant biotech & Risk Assessment

McBride, Timothy [1], Fuentes, Cesar [2], VanWinkle-Swift, Karen [2].

Using disruption of a resident gene as the primary step in screening for transformation of Chlamydomonas monoica with foreign DNA.

Sexual reproduction in Chlamydomonas monoica leads to the production of a heavily walled, highly resistant zygospore. Classical genetic analysis of mutant strains has led to the identification of many genes important for the unique resistance properties of the zygospore. A first step toward determining the function of these genes is to develop a protocol for successful transformation of C. monoica cells with exogenous DNA. We have used two cell wall-less strains of C. monoica as recipients and have introduced a foreign gene (aphVIII) conferring resistance to the antibiotic paromomycin. Uptake of the aphVIII gene was enhanced through the use of electroporation or vortexing cells with glass beads in the presence of polyethylene glycol . We anticipated poor expression of the foreign gene (due to endogenous gene silencing and because the foreign gene was driven by C. reinhardtii promoters). Therefore we avoided using a primary screen for transformation that would require foreign gene expression. Instead the primary screen was for chlorate resistance which could result from insertion of the foreign DNA into any of several resident genes involved in nitrate metabolism. Chlorate resistant colonies were then transferred to non-selective medium for recovery and further growth. This increases the probability of detecting low level expression of the inserted aphVIII gene when the cells are subsequently plated on a paromomycin-containing medium. DNA was extracted from a total of six transformants showing weak resistance to paromomycin, and integration of the foreign gene was confirmed by PCR using aphVIII specific primers. Supported by NIH grant 1R15GM071374-01.


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1 - Northern Arizona University, Biological Sciences, Box 5640, Flagstaff, AZ, 86011-5640, USA
2 - Northern Arizona Universtiy, Biological Sciences

Keywords:
Chlamydomonas monoica
transformation
aphVIII.

Presentation Type: Plant Biology Abstract
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P45031
Abstract ID:2633


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