Pcdna3.1 expression vector

The C-terminal FLAG-tagged mouse mGluR2 construct in pcDNA3.1(+) expression vector was purchased from GenScript (ORF clone: OMu19627D) and verified by sequencing (ACGT Inc). Full length mGluR2 construct with an amber codon (TAG) mutation of amino acid A548 (azi-CRD) or N-terminal SNAP-tag (SNAP-mGluR2) were generated as previously reported (Liauw et al., 2021 (link)). The insertion of an amber codon (TAG) between E715 and V716 in mGluR2 (azi-ECL2) was performed using the QuikChange site-directed mutagenesis kit (Agilent). SNAP-mGluR2 constructs used for calcium imaging had C-terminal FLAG-tag removed by PCR-based deletion using phosphorylated primers. All plasmids were sequence verified (ACGT Inc). DNA restriction enzymes, DNA polymerase and DNA ligase were from New England Biolabs. Plasmid preparation kits were purchased from Macherey-Nagel.

A construct containing the ORF of human CPE-QQ was custom synthesized and cloned into the pcDNA3.1(+) expression vector with BamH1/Xho1 cloning sites (GenScript, Piscataway, NJ, USA). Sequence analysis confirmed the insertion of three adenosine nucleotides consistent with the EST database sequence of the mutant hCPE (Genbank ID# DA134138). The pcDNA3.1 vector was digested with NruI/PvuII to generate a 2.44 kb fragment containing the CMV enhancer and promoter, the CPE-QQ ORF and the vector poly A site. This fragment was purified and used for transgenic mouse production by Dr James Pickle (Transgenic Core Facility, NIMH, NIH) on a C57/BL6 background. Founder animals were genotyped with two sets of primers; QQ1 5′-CCGCGTTACATAACTTACGGTAAATGGCC-3′, QQ2 5′-GGGTCTCCCTATAGTGAGTCGTATTAATTTCG-3′ that amplifies the 5′-upstream sequence of the pcDNA3.1 vector only; and QQ4 5′-GCTTGCTCCTGAGACCAAGGCTG-3′, QQ6 5′-GCCTGCTATTGTCTTCCCAATCCTCC-3′ that amplifies the 3′-end of hCPE and the downstream pcDNA3.1 vector. Positive founders were mated with C57/BL6 WT mice to propagate the lines, and F1 and subsequent generations were genotyped for the transgene identification. All animal studies were done in accordance with the NICHD Animal Care and Use Committee guidelines.

A mouse CaSR construct with a C-terminal FLAG-tag in pcDNA3.1⁺ expression vector was purchased from GenScript (ORF clone: OMu14241D) and validated by sequencing (ACGT). A SNAP-tag (pSNAP_f, NEB) flanked by GGS linkers was inserted at position 21 using HiFi DNA Assembly Master Mix (NEB). Point mutations in CaSR (P55L, C129S, C131G, C129G + C131G, T151M, L174R, R220W, R227L, E228K, G557E) were introduced using a QuikChange site-directed mutagenesis kit (Qiagen). The SNAP-tag CaSR construct was used as the template for mutation of amino acid E593 to an amber codon (TAG) via QuikChange mutagenesis. AscI restriction sites were used to insert mEGFP (mEGFP-N1, gift from Michael Davidson (Addgene plasmid # 54767) at the C-terminus of the GenScript construct, and it was used as the template for mutation of amino acid D451 to an amber codon (TAG) via QuikChange mutagenesis. All plasmids were verified by sequencing (ACGT). DNA restriction enzymes, DNA polymerase, and DNA ligase were purchased from New England Biolabs. Plasmid preparation kits were obtained from Macherey-Nagel.