Primers for validation of Rosa-Gpr56 mice

Three groups of primers were used for PCR screening of the RosaGPR56 mice, as detailed in our citation #60 (Chu et al., 2016). The primers are as follows:

1. Validation of Recombination in Rosa26 Locus:
   • R26F2: GCCTCCTGGCTTCTGAGGACCG
   • R26R2: TCTGTGGGAAGTCTTGTCCCTCC
   • SAR: CCTGGACTACTGCGCCCTACAGA
2. Validation of Target Gene Insertion (HA-GPR56):
   • GPR56 HA F: GATTACGCTCCCCGAGAAGACTTCCGCTTCTG
   • GPR56 R: TTCTCGGGGGAGCGTAATCTGGAACATCGTATGG
3. Confirmation of Integration of Insertion:
   • R26R3: CTGCCCGAGCGGAAACGCCACTGAC
   • SAR: CCTGGACTACTGCGCCCTACAGA

All PCR products of mutation/insertion bands were later Sanger sequenced using one of the PCR primers. Further validation was done by Sanger sequencing of the entire insertion of the GPR56 gene using PCR products with the following primers:

• Primer F: ttatacgaagttatcggcgcgccATGGCTGTCCAGGTGCTGCG
• Primer R: ATTGATCGCGGCCGCGGCGCGCCttagatgcggctggaggaggtg

(Note: The uppercase base letters in these long primers are from HA-GPR56 cDNA, and the lowercase base letters are from the pR26 CGA/GFP ASCI vector sequence. This confirms the integration of the GPR56 cDNA insertion, though it may not apply to other transgenes.)

All PCR reactions were conducted with an annealing temperature of 60°C or according to the instructed annealing temperature from Integrated DNA Technologies. In cases of high GC content, 5% DMSO or other GC-rich PCR kits were used. High-fidelity PCR kits might be applied for products used for sanger sequencing.

The transgenic mice were further confirmed by immunohistochemistry and RT-qPCR, as shown in the manuscript. Given the increasing affordability of whole genome sequencing, it is recommended to conduct whole genome sequencing to validate the insertion of target genes and rule out off-target effects.