Project director: Sharon Isern

As genes and promoter elements from Daphnia are identified, it will become increasingly important to have the ability to manipulate them and reintroduce them in Daphnia to study their function. Our current efforts include the development of transformation systems including physical methods, viral vectors, and transposon-based systems. To address the challenges of delivering transgenes and their expressions inside the cell, we are exploring the use of electroporation and particle bombardment, and viral vectors that have the innate ability to efficiently introduce their own genetic material into host cells. Depending on the transformation system used, transgene expression in Daphnia cells will either be transient or stable. For the viral vector systems, the nature of the parent virus (whether it integrates its genome as part of its lifecycle) will determine if expression of the transgene will be long-term. We are also exploring the use of heterologous and homologous (Pokey) transposon-based vectors in Daphnia transformation. A transposon is a small DNA element that bears short inverted repeats at its ends, and includes a single gene that encodes the enzyme, transposase. The transposase enzyme recognizes the inverted repeats of the transposon and catalyzes its excision and insertion into the host cell genome. Pertinently, transposons can be adapted to serve as tools for introducing genes into cells. As proof of concept for transformation using the various systems, we will be incorporating transgenes containing reporter genes driven by heterologous and Daphnia-specific promoter elements.

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