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Các plasmid được tiêu hóa với các enzyme hạn chế thích hợp và chèn được tinh chế bằng điện di agarose gel. Các mảnh vỡ DNA tuyến tính được microinjected vào pronuclei trứng thụ tinh chuột C57BL / 6SnJ sau đó được reimplanted vào ống dẫn trứng của bà mẹ nuôi CD1 pseudopregnant (Phòng thí nghiệm Jackson). | 72 Takeda et al. Fig. 11 . Schematic representation of the roles of Cbfal in endochondral ossification. Cbfal favors chondrocyte hypertrophy via an Ihh-dependent pathway. In turn Ihh induces differentiation of the cells of the bone collar through a Cbfa1-dependent pathway. Cbfa1 also favors VEGF expression. Generation of Transgenic Mice Plasmids were digested with appropriate restriction enzymes and inserts were purified by agarose gel electrophoresis. Linear DNA fragments were microinjected into pronuclei of fertilized C57BL 6SnJ mouse oocytes that were subsequently reimplanted into oviducts of pseudopregnant CD1 foster mothers Jackson Laboratories . al II Cbfal a 1 II Cbfala and aI II CbfalAPST transgenes were respectively coinjected with the 1.3kb of OG2-LacZ construct to obtain transgenic mice coexpressing the two transgenes. Genotypes were determined by polymerase chain reaction PCR using the following as primers 5 -GGCAGCACGCTATTAAaTccAa-3 and 5 -GGTTTCAGGGGGaGgtGTG GGaGg-3 for the al II Cbfal mice 5 -CTGGACATCATAGCAAAGGCCC-3 and 5 -GGTTTCAG GGGGAGGTGTGGGAGG-3 for the al II Cbfala mice and 5 -CGGAGCGGACGAGGCAAGA GTTTC-3 and 5 -GGTTTCAGGGGGAGGTGTGGGAGG-3 for the al II CbfalAPST mice. Sex was determined by PCR using the Ary-specific primers 5 -CATGACCACCACCACCACCAA-3 and 5 -TC ATGAGAcTgCCAAcCaCAG-3 25 . Reverse Transcription PCR Analysis To monitor the transgene expression total RNA was prepared from 12.5-dpc embryos. Three to four embryos were analyzed independently for each genotypes. RNA extraction cDNA synthesis and PCR amplification were performed using standard protocols 26 . Exon 2 amplification of the HPRT gene was used as internal control of the quantity and quality of the cDNAs. The following sets of the primers were used transgene specific PCR 5 -CCAGGCAGTTCCCAAGCATT-3 and 5 -AGAG CTAtGaCGTCGCATGCAcAc-3 endogenous Cbfal 5 -GGCAGCACGCTATTAAATCCAAA-3 and 5 -TGACTGCCCCCACCCTCTTAG-3 and Hprt 5 -GTTGAGAGATCATCTCCACC-3 and 5 -AGC GATGATGAACCAGGTTA-3 . .
