PCR Genotyping
PCR genotyping still needs to be performed to ensure proper insertion of the F1 replicon cassette into the Tn7 modified BAC. It is expected from PCR genotyping using the same primer pairs from before; there should be differences in Fragments 11 and 12, as well as Fragment 6 from the Tn7 insertion site insertion.
Whole Genome Sequencing
To ensure the integrity of our construct and to determine if any point mutations have occurred that may inhibit the viral vector for use; whole genome sequencing is necessary. Additionally, sequencing of all 3 BAC used thought the construction of the final product will be performed.
Comparison between other viral vector systems
One test that can be performed is a blue/white selection test in order to determine the transformation efficiency of our design. In addition, a comparison test between other viral vector systems can be performed to determine the ease of insertion of a transgene and the possible insertion size.
For Future Applications as a Viral Vector
First, a fluorescence reporter gene is should be added to determine transgene expression capabilities and if any optimizations are needed beforehand. In addition, the F1 replicon should be removed if possible for added ease of use in academic research. One would need to characterize the toxicity of the viral vector in vitro using a cell assay to ensure its safety and efficiency for use in an academic setting. In addition, before any repetitive use, a viral titer is needed to determine the minimum dosage needed for transgene expression.
Leader: Wasu Ngamkanjanarat