Total RNA was isolated from transgenic mouse liver, kidney and small intestine by using TrizolH method (GIBCO-BRL/ Invitrogen Corp., Carlsbad, CA). Briefly, samples were homogenized in 800 ìL TrizolH (GIBCO-BRL/Invitrogen Corp., Carlsbad, CA), followed by the addition of 160 ìL chloroform. After centrifugation at 4uC at 14,000 rpm for 15 min, the top layer was transferred to a clean microcentrifuge tube and 400 ìL of 2propanol was added, incubated at room temperature for 20 min and centrifuged at 14,000 rpm for 10 min at 4uC. Total RNA was washed in 75% ice-cold ethanol once and after 5 min was centrifuged at 14,000 rpm at 4uC. RNA pellets were air dried and dissolved in 50 ìL RNase-free water. Complementary DNA (cDNA) was prepared using the OmniscriptH reverse transcription system (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. The reverse transcription system contains oligo primers, RNase inhibitor, MMLV reverse transcriptase, dNTP mix, buffer and 3 ìg total RNA in a final volume of 20 ìL. The reaction mixture was incubated at 42uC for 1 h. CEACAM1 isoforms were detected by PCR on a Bio-Rad iCycler PCR (Bio-Rad Laboratory, Hercules, CA) with 2X fideliTaq PCR master mix (USB, Cleveland, Ohio). cDNA (5 ml) was added to a final 25 mL volume reaction. The sense strand primer 59-AACGT-CACCCAGAATGAC-39 and antisense strand primer 59-CTCAG-GACCACTCCAATGA-39 target human CEACAM1 gene. The amplified products are 1118 bp (4L), 1066 bp (4S), 831 bp (3L), 779 bp (3S). The amplification parameters were initiated at 95uC for 5 min, then 95u C for 30 s, 55uC for 90 s, and 72uC for 30 s for 38 cycles, followed by 7 min at 72uC for the final extension. For mouse CEACAM1, the sense strand primer 59-GA-CTCTGTCGACGACATGGAGCTGGCCTCAGCACATC-39 and antisense strand primer 59-GACTCTATCGATTCACTTC-TTTTTTACTTCTGAATAAAC-39 were used to PCR amplify the murine Ceacam1 cDNAs. The four RT-PCR amplified products for murine Ceacam1 correspond to the 4L, 4S, 2L and 2S mRNA splice isoforms. The amplification parameters were same as above.