Pfuse higg1 fc

The vector used to express cell-surface protein ECDs (Figure 2A) was generated by Gibson cloning and adapted from the commercially available pFUSE-hIgG1-Fc (InvivoGen) vector. Each ECD was subcloned between an N-terminal IL2 Signal Sequence and a C-terminal TEV cleavage site.
BirA was subcloned into a pLX302 lentiviral vector with a C-terminal KDEL ER localization tag.
We used a previously described vector for expression of Fabs (Hornsby et al., 2015 (link)). The pFUSE-hIgG1-Fc (InvivoGen) vector was used for expression of IgGs wherein the heavy chain was genetically fused to the hIgG1-Fc and the light chain was expressed on a separate copy of the vector with a C-terminal FLAG tag. The pFUSE-hIgG1-Fc (InvivoGen) vector was also used for expression of the αCD19- αCDCP1. The αCD19 was C-terminally fused to the light chain of the αCDCP1 Fab and the heavy chain was expressed on a separate copy of the vector.
Previously described vectors were used for CRISPRi experiments. Individual sgRNAs were cloned into a pU6 lentiviral vector (Adgene: 46914), dCas9-BFP-KRAB was expressed from a pHR-SFFV lentiviral vector (Adgene: 46911), and the sgRNA library was cloned into a pSICO lentiviral vector (Adgene: 84832).

To generate a construct expressing recombinant proteins consisting of human IgG Fc and extracellular domains of Siglec-G or H, the corresponding cDNA fragment was amplified by PCR and subcloned into expression vector pFUSE-hIgG1-Fc (Invivogen). cDNA for Neu1-4 were amplified by RT-PCR and subcloned into expression vector pCDNA6 (Life technologies, Grand Island, NY). All constructs were verified by restriction enzyme digestion and DNA sequencing. For purification of Siglec-G-Fc or Siglec-H-Fc, the corresponding expression vector was co-transfected with a GFP expression vector into 293 T cells, and stable clones were obtained after 2 weeks culture in selection medium containing 2.5 μg/ml puromycin and 50 μg/ml Zeocin. The stable clones were amplified and cultured in serum free medium, Siglec-G-Fc or Siglec H-Fc was purified with a protein A column from the cell culture supernatants.