Hypoxanthine aminopterin thymidine supplemented medium

Nucleic acid gel imaging system, nucleic acid electrophoresis apparatus and protein gel electrophoresis apparatus were purchased from Shanghai Tanon company. Microplate reader was purchased from Thermo Fisher Technology Ltd (Shanghai, China). Polyethylene glycol(PEG) was purchased from Merck Co, Mouse typerisotyping panel kit from Bio-RAD Co, and RPMI MEDIEM 1640 medium, penicillin-Streptomycin double antibody solution, newborn calf serum and HEPES from Life Technologies Gibco Co. Hypoxanthine-Aminopterin-Thymidine (HAT) supplemented medium, Hypoxanthine- Thymidine (HT) supplemented medium, Freund's complete or incomplete adjutants were purchased from Sigma. Natural human cystatin C (N-HCC) was purchased from Enzo Life Sciences Ltd., Horseradish peroxidase-conjugated goat anti-mouse IgG from Santa Cruz Biotechnology Inc., or Tween-20 and Bovine serum albumin (BSA) from Amresco. BALB/c mice were from Shanghai Institutes for Biological Nutrition, according to the ethical permission approved by the committee of Animal Ethical Evaluation, Chinese Academy of Science. The natural camel single-domain heavy chain antibody library was kindly provided by Dr. Ario de Marco for Italian IFOM-IEO center.

2 × 10⁶ CHO(DYS‐HAC2) cells were co‐transfected with p17 (8 μg) and Cre‐expressing plasmids (1 μg) using Lipofectamine 2000 (Invitrogen) to induce insertion of p17 into DYS‐HAC2 via Cre/LoxP system and generate DYS‐HAC3. 24 h after transfection, CHO cells were expanded with a hypoxanthine–aminopterin–thymidine (HAT)‐supplemented medium (Sigma‐Aldrich) to select only CHO cells containing DYS‐HAC3. CHO(DYS‐HAC3) cells were further transfected with pP‐ΔHR and Bxb1‐expressing plasmid following the same protocol detailed above. Selection was performed according to the following: 8 μg of blasticidin S (Wako), 800 μg of G418 (Promega), 7 mM of L‐histidinol dihydrochloride (Sigma‐Aldrich) and HAT medium.