| This study was conducted to establish an in vitro model for culture of lactating bovine mammary tissue. Comparison was firstly made of cultured methods for mammary tissue in vitro (Expt. 1). The conditions for amplification of asl casein and GAPDH gene from mammary tissue of lactating dairy cows were then established (Expt. 2). The activity of the cultured mammary tissue from lactating dairy cows was examined in terms of the response in the expression of as1 casein gene to prolactin (Expt. 3) and to methionine (Expt. 4), respectively.1. Comparative research of cultured methods for mammary tissue of lactating dairy cows in vitro.Mammary tissue from dairy cows was cultured in roller tube and stationary culture (24 well plastic culture plate). Explants were immediately incubated at 37℃ (95% air; 5% CO2) in DMEM/F,2 medium (4-ml volume) containing 10% FCS, 2μg/ml hydrocortisone, l0μg/ml insulin and 5μg/ml transferrin. In roller tube culture, the tissue still maintained the activity at 72h, which was indicated by the value of MTT and oligonucleosomal fraction of cellular DNA. While in stationary culture, the mammary tissue did not attach entirely to the bottom of culture plate until 24h. The droplet of fat was secreted from cultured mammary tissue in 72h. Through the value of MTT and oligonucleosomal fraction of cellular DNA, it was demonstrated that the53mammary tissue proliferated from 72h, and the apoptosis rate of cultured tissue was declined markedly. The proliferated ability and apoptosis rate of cultured mammary gland was stable from 96 to 144h.Both the roller tube and stationary methods could maintain the basic active state of the cultured tissue. The roller tube culture had the advantages of the simple operation procedure and the short period of experiment. And the amount of cultured tissue was more than that of stationary culture. However, it was inconvenient to observe the cultured tissue and take photography in this method, and it was also difficult to observe the proliferation and secretion of cultured tissue. Contrasted with the roller tube culture, the mammary tissue in stationary culture could keep the active state for longer time and could be observed and taken pictures at any times. Therefore it was convenient to study the morphology of the cultured tissue. The disadvantage of the stationary culture was that it required time to insure the tissue to attach to the bottom of plastic culture plate, resulting a prolonged period of experiment.2. RT-PCR conditions for a_s1 casein and GAPDH gene from mammary tissue of dairy cows.Total RNA was extracted from mammary tissue of lactating dairy cows. According to the asi casein gene and housekeeping gene GAPDH sequence reported previously, their upper and lower primers were designed. Partial cDNA fragment in length 434 and 584bp were synthesized and amplified by RT-PCR. The PCR products were then sequenced. It was demonstrated that the fragments lengths were the exactly same as the designed respectively, and 130 amino acid residues were coded by the fragment of asi casein, which were the major part of ctsi casein mature peptide. Homologous analysis showed that the fragment of asi casein was consistent with the reported bovine asi casein cDNA sequence, except that the G was changed to C at the 251st bp. The fragment of GAPDH was consistent entirely with the reported dairy cows GAPDH cDNA sequence.The different MgCk concentration, cycle degree and the suitable primers concentration in PCR system were investigated. The optimal MgCk concentration, cycle degree and the primers concentration were observed at l.0mmol/L, 30, and 0.4umol/l for asi casein, and were at 1.5mmol/L, 30, 0.6urnol/l for GAPDH, respectively. PCR were conducted separately.543. Effect of prolactin on expression of asl casein determined by semiquantitative RT-PCR analysis in cultured mammary tissue from lactating dairy cows.The mammary tissues from lactating dairy cows were incubated at 37癈 (95% air; 5% CO:) in DMEM/Fi2 medium (4-ml volume) containing... |
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