AccuPrime Pfx DNA polymerase (Invitrogen) was used for site directed mutagenesis using the QuikChange II Site-directed Mutagenesis protocol from Agilent Technologies. The following murine stem cell virus (MSCV)-based retroviral vectors were used to express Unc93b1, TLR9, TLR7, and TLR3 in cell lines: MSCV-PuromCherry (IRES-PuromycinR-T2A-mCherry), MSCV2.2 (IRES-GFP), MSCV-Thy1.1 (IRES-Thy1.1), and MIGR2 (IRES-hCD2). 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK) was fused to the C-terminus of Unc93b1. TLR9, TLR7, and TLR3 were fused to HA (YPYDVPDYA) at the C-terminal end. TLR7 sequence was synthesized after codon optimization by Invitrogen’s GeneArt Gene Synthesis service as previously described10 (link). TLR9 chimeras of the juxtamembrane and transmembrane regions were previously described4 (link).
Protocol detail
Expressing TLRs and Unc93b1 in Cell Lines
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AccuPrime Pfx DNA polymerase
QuikChange II Site-directed Mutagenesis protocol
GeneArt Gene Synthesis service
Cell lines
Chimeras
Codon
Dna polymerase
Gene service
Ires
Murine
Retroviral
Site directed mutagenesis
Stem cell
Synthesis
Unc93b1
Vectors
Virus
Corresponding Organization :
Other organizations : University of California, Berkeley
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Variable analysis
independent variables
- Expression of Unc93b1, TLR9, TLR7, and TLR3 in cell lines
- Fusion of 3xFLAG to the C-terminus of Unc93b1
- Fusion of HA to the C-terminus of TLR9, TLR7, and TLR3
- Codon optimization of TLR7 sequence
- Not explicitly mentioned
- Not explicitly mentioned
- Positive control: Not specified
- Negative control: Not specified
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