Creation of human CTLA4 knock-in mice. (A) Schematic diagram of the structure of construct. The primer positions for screening the floxed and deleted genotypes are also illustrated. PCR Reaction A used primers outside the loxP sites spanning the Neo/TK gene. A successful excision (deleted) of Neo/TK produced a 1.1-kb fragment, whereas undeleted (floxed) Neo/TK did not produce a fragment due to the PCR conditions used. PCR Reaction B used a forward primer outside of and a reverse primer within the Neo/TK cassette. (B) Southern blot of DNA from ES cells transfected with the human CTLA4 construct. A 7-kb band represents successful homologous recombination with the human CTLA4 construct, whereas a 4.7-kb band represents an unaltered mouse Ctla4 gene. (C) Excision of Neo/TK by Cre-recombinase. As depicted schematically in panel A, Reaction A produced the expected 1.1-kb fragment, whereas Reaction B amplified no fragment, consistent with successful deletion of Neo/TK. (D) Expression of human and mouse CTLA-4 RNA in homozygous (left panel) and heterozygous (right panel) knock-in mice. Spleen cells from human CTLA4^+/– and human CTLA4^+/+ mice were stimulated for 30 hours in vitro with 0.1 μg/mL anti-CD3 mAb 2C11. RNA was extracted and RT-PCR was performed. Primers spanning the full-length CTLA4 RNA sequence were used to confirm that full-length RNA of the knock-in gene was being expressed (left reaction), whereas those that were specific for either mouse (mE2) or human (hE2) CTLA-4 exon 2 were used to identify mouse and human CTLA4, respectively.