Matchmarkergal4 two hybrid system 3

MatchmarkerGAL4 Two‐Hybrid System 3 (Clontech Laboratories, Mountain View, CA) was used for screening. The bait plasmid contains the Shox2a coding sequences without its transactivation domain (C‐terminal 19 amino acids) in pGBKT7. For library screening, bail plasmid was transformed into yeast strain AH109. The transformed yeast was cultured and mated with the Matchmaker pretransformed 11‐day mEmbryo cDNA library (Catalog No.: 638868; Clontech Laboratories). Yeast colonies were selected on yeast nitrogen base medium that contains 2% glucose and lacks adenine, histidine, leucine, and trytophan. Approximately 1.15×10⁷ cotransformants were obtained. Colonies were picked and checked for beta‐galactosidase (β‐Gal) production using a filter assay with 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside. Plasmids were isolated from positive clones, and a second round of interaction screening was performed to confirm the interactions. The inserts from positive clones were sequenced.