Troubleshooting Diagram Lentis
Contents
1. transduction Check vector copy number by QPCR Presence of HIV sequences Absence of HIV sequences in target cell genome in target cell genome Vector contains a Vector contains a ya si Check genomic RNA tissue specific promoter specific gene Check length of integrant possible splicing of gene Genomic RNA is absent Genomic RNA is present in Check gene expession in vector particles vector particles Western blot Immunofluorescence Transduce specific cell type with a promoter GFP vector Defect in reverse transcription or integration No GFP expression GFP expression Defect in packaging in specific target cells in specific target cells of genomic RNA Possible causes Check length of integrant Possible causes vossible splicing of gene Check gene of interest Packaging signal renaire Check strength of polyadenylation promoter maturation in PPTs or PBS transfection in cells mutations in int sequence integrity of open reading removed during cloning frame no ATG before gene or vector production ATG etc Genomic RNA is not produced Reclone vector Reclone vector
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