Matt Gethers/20.380 HIV Project/Technical Paper/In Vitro Tests.doc

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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