| l.A total of 312 fetuses were collected from 27 WuZhiShan inbreeding Pigs (WZSP) and White Minitype Pigs to study the factors influencing the establishment of Porcine Embryonic Germ cells, and we draw the following conclusions:(1 )We can get embryonic fibroblasts from d26-28 fetuses (day of first mating is day 0, that is d 0); In this thesis, STO and MEF1 were inactivated by incubation in medium containing 10 g/mL of mitomycin C for 1.75h~2h; While PEF were inactivated by the same medium for 2.25h~2.5h.(2) Fetuses at d27 were collected from WZSP and White Minitype Pigs respectively, the results showed that there is no notable difference in EG cells culturing between the two types of pigs (P> 0.05) .they are all suitable materials to establish porcine EG cell lines.(3) When the fetal stage was considered, the results showed that under the same conditions, there is no notable difference in EG cells culturing using fetuses collected on d26, d27, d28, respectively (P >0.05) .(4) PGCs collected from d27 were cultured in two different medium [Medium 1: DMEM + sodium pyruvate + 15%FCS (fetal calf serum) + 1% L-glutamine + 1%MEM Non-Essential Amino Acids Solution + l%Penicillin-Streptomycin + lOuM (3 -mercaptoethanol; Medium 2: DMEM: HF10 (V:V=50:50) + sodium pyruvate + 15%FCS + 1% L-glutamine + 1% MEM Non-Essential Amino Acids Solution + l%Penicillin-Streptomycin + lOuM P -mercaptoethanol + lOOOIU/mL LIF + 40ng/mL SCF + 20ng/mL bFGF]. The results showed that there is no notable difference in EG cells culturing in these two medium (P>0.05) .(5 ) When PGCs were cultured on different feeder layers, the better results were got if PGCs were cultured on STO and MEF1 feeder layers, When PGCs were cultured on thawed STO feeders, PEF, inactivated PEF feeders, 0.1% gelatin, we can not get typical EG colonies.(6) PGCs were obtained from fetal gonads either by mechanical procedures or by mechanical procedures with prior 0.02% EDTA treatment, the results showed that there is no difference in obtaining porcine EG cell colonies if the two PGCs collecting methods were used(P>0.05); We also tested PGCs seeding density, the results showed PGCs from 1 fetus seeded in 2 or 3 wells of a 4 -well plate is more suitable than seeded in 1 or 4 wells of a 4 -well plate.( 7 ) In order to establish a feeeder-free system in porcine EG culture, we put PGCs on STO feeder layer, on STO extracellular matrix or on 0.1% gelatin in BRL-CM, and the results showed that when PGCs cultured on STO feeder, the BRL-CM had a positive effect on keeping the undifferent state of EG colonies. But we got few colonies when PGCs were cultured on STO extracellular matrix or on 0.1% gelatin even in BRL-CM.(8) With the support of STO feeders, addition of growth factors is not reqired for survival and proliferation of curtured porcine PGCs (P>0.05) . Although the growth factors did have a positive effect on keeping the undifferent state of EG colonies cultured on STO feeders, we can not get EG colonies when PGCs were cultured on 0.1% gelatin in medium supplemented with LIF, SCF and bFGF.(9) The primary EG colonies were passed on d2, d3, d4, d5, d6, d7 respectively after large quantities of EG colonies were found (day of first finding large quantities of EG colonies is day 1), the results showed the primary EG colonies should be passed on d2-4; When the primary EG colonies were passed, three methods were used: a, "0.2%Trypsion-0.04%EDTA" + "the once dispersed method"; b, "0.2%Trypsion-0.04%EDTA +1% chicken serum" + "the once dispersed method"; c,"0.2%Trypsion-0.04%EDTA +1% chicken serum"+ "the series dispersed method", the results showed the three methods had a equally effect on the passage of porcine pimary EG colonies.2.Three kinds of cells were used for microinjection. a, PGCs collected from fetuses were immediately injected into the embryos after isolation; b, PGCs were cultured on gelatin-coated multidishes for up to 24 h untill injection; c, EG cells of 0-6 passages. We got 564 embryos from 42 (Large White X ChangFeng White) pigs, about...
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