Matt Gethers/20.380 HIV Project/Technical Paper/In Vitro Tests.doc
In Vitro Tests
- Genomic Testing of Stem cells
- PCR, RT-PCR to see if things get added and expressed
- Proteomic Testing of Stem cells
- Yes siRNA
- No RNAses, CD4+
- Genomic Testing of RBCs
- Aka we want to see nothing in an RT-PCR or PCR
- Proteomic Testing of RBCs
- Look for CD4+ (how much?), proteases
- Make sure the Stem cells only turn into RBCs
- Test by adding media that should trigger other differentiation
- Death Switch
- Make sure our ‘death switch’ will kill only our RBCs/HSCs, not normal RBCs/HSCs
- Determine concentration necessary to kill cells with death switch (whoot cytotoxicity)
- Functioning of RBCs
- Deformation tests – indicative of clearing from blood
- Still able to carry oxygen?
- HIV Infection controlled
- See if the HSCs get infected (two fold: 1 see if they get infected, 2 see if the siRNAs work)
- See if the RBCs get infected
- How many virions per cell
- Under normal conditions
- If at max capacity of virions, do cells explode
- Is the virion released able to infect?
- Do the virions get destroyed
- Is it by siRNA or RNAses primarily?
- Put engineered RBCs and T cells together
- Who gets infected?
- How many RBCs are necessary per T cell
- How many eRBCs versus normal RBCs?
- If RBCs filled with virus (but no free floating virus) are added to T cell culture, do T cells get infected?
- Do we see resistance in HIV?
- Look for evolution of strain