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
 * Differentiation
 * 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?
 * Co-culture
 * 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?

HIV effects

 * Do we see resistance in HIV?
 * Look for evolution of strain