Our goal is to engineer cells to express a synthetic receptor under the control of an endogenous promotor. We will therefore perform a CRISPR-Cas9 knock out (using RNPs) of the endogenous gene (targeted to the non-coding region of the first exon) and supply a HDR template with homology arms (lenght needed?). The HDR template will be transduced with a AAV and bears two cargos: the synthetic receptor (Kozac + signal peptide + myc tag + ORF) and a response element (Gal4 UAS + mCMV) with its target (Kozac + Cytokine ORF + yet to be defined tag OR P2A + mCherry/BFP...).
My questions in particular are: How much space would you leave between the receptor sequence and the response element for it to work? Are there any spacial requirements or additional motifs needed for efficient transcription/translation of both the receptor (by the endogenous promotor) and the Cytokine (by the Gal4 UAS + mCMV)?
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