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