23. Since the DNA is not integrated into the genome of the host, these genes STOP replicating prior to cell division and subsequently are lost to the daughter cells Will require re-administration so that descendant cells will express gene of interest CELL VECTOR NUCLEUS http://www.virobathe.org/M055250-Adenovirus-SPL.jpg
46. Protein levels downregulate disproportionately in comparison to intracellular markers (e.g. GFP) following transfection and may subsequently not reflect actual levels of secreted transgene product
47.
48. Gene expression was dependent upon viral titer
64. Expression Assays: Following homogenization, run a gel and perform a WB OR PCR amplify using engineered primers Northern/ Western Blots * 25 L of 1 x 108 TU/mL into the right gastrocnemius Southern Blots
Doxycycline = tetracycline type antibioticTetracycline inducible systemTheoretically it should be possible to switch the expression on/off depending on the presence of doxycyclineMethod:Transfect cells (e.g. schwann cells) with gene of interest, add factor required for “on”
EYFP = Enhanced Yellow Fluorescent ProteinIpsilateral = on the same sideContralateral = on the opposite side
~ 300- 400 neurons that correspond to the area and since 32- 43 neurons were found to be EYFP + ~10% of motor neurons can be transducedths way using the titer levels as outlined by thegroupUsing a higher concentration of virus may not be the best mechanism as the immune system may have a more pronounced effect
+ EYFP staining only seen in a subset of neurons since these are the once that are innervating the muscle of the injected site!!!!
