1 compatability test in hepatocytes(in progress)

One of the aims of our project is to build a pathway that can be sustained in iPSC-derived hepatocytes and remains functional to eliminate spontaneous cancerous cells. As proven previously, miR-122 targeted suicide pathway will be knock down in cells with high level of miR-122 (in our test, miR-122 level as high as… is adequate to knockdown …percent of gene expression which is far more than enough to function in hepatocytes with endogenous miR-122). We still need to confirm this result in hepatocytes.

Mouse primary hepatocytes are successfully separated and then infected with our pathway by lentivirus. Since primary hepatocytes is difficult to be transfected, we have used lentivirus under ultracentrifugation to increase the efficiency of integration. The design of our pathway in lentivirus is described in …page. Generally, it takes 2 weeks to complete selecting for a constant expression cell line. Because our tet-off system resides in 2 independent plasmids, it takes additional time to finish the successive antibiotic selection for both 2 modules.

We still need to test whether this pathway will interfere the normal function of hepatocytes by doing…

The Cruel Guard：Suicide Gene and Its Ancillary Facility

1.Comparison between Suicide Genes: who is the most tough killer?

We collected several different Suicide Genes which functions in different pathways and patterns. In order to choose one that is most capable of inducing apoptosis in cancer cell, we first carried out a comparison experiment for these genes.

2 pluripotent test in iPSCs( in progress)

Our pathway is designed to be inplanted in the iPSC-induction phase and begin to function after redifferentiation. It should reach the following standards:

1. promoter is functional during the time of redifferentian
2. leakage expression is tolerable
3. pluripotent remains in iPSCs with pathway
4. directional redifferention should be increased
5. our pathway can kill transformed cancerous cell
6. our pathway is functional in long term

3 fine-tuning of parts

4 directional differention test in iPSC

5 tumorgensis test in iPSCs

Discussion

Include several standard questions and answers frequently asked.