ABSTRACT
To improve the transfection efficiency of chicken primordial germ cells (PGCs), the present study evaluated the plasmid dosage and cell number on the efficiencies of three transfection reagents (Lipofectamine 2000, 3000 and LTX & Plus Reagent). PGCs was isolated from embryonic gonads of Huiyang bearded chicken. After 60 days of culture in vitro, the cells were transfected by using Lipofectamine transfection reagents with piggyBac vectors coding for the green fluorescence protein (GFP). PGCs were passaged in culture and fluorescent cells were screened and selected by flow cytometry at three days after transfection. At three weeks post transfection, about 2000 cells were injected into the stage 16 Hamburger and Hamilton (HH) embryos and incubated until stage 30 HH. The results showed that Lipofectamine 3000 was the best for transfection of PGCs. The highest transfection efficiency of PGCs could be achieved with a combination of 3 µg plasmid, 4 µL Lipofectamine 3000 transfection reagent and 0.5×10 4PGCs cells. Flow cytometry analysis showed a 23.4% efficiency of stable transfection of PGCs using Lipofectamine 3000 with piggyBac vector, which was improved 2 times or more over current commonly used methods. After reinjecting PGCs into recipient chicken embryos, GFP-positive cells were observed in the gonads of the recipient chicken embryo by fluorescence microscopy. The study comprehensively evaluated the factors of transfection reagents, plasmid dosage and cell number to optimize the transfection of PGCs, thereby providing a foundation for the efficient preparation of transgenic and gene-edited chickens.
Subject(s)
Chickens , Germ Cells , Animals , Animals, Genetically Modified , Chick Embryo , Chickens/genetics , Gonads , TransfectionABSTRACT
Objective To explore the proliferation-promoting effect of bovine mammary gland epithelial cells (BMECs) co-cultured with umbilical cord mesenchymal stem cells (UC-MSCs) in serum-free culture mediuum.Methods Bovine UC-MSCs and BMECs were selected for co-culturing in direct or indirect contact.In the direct contact culture groups, UC-MSCs and BMECs were co-cultured at concentration ratios of 2∶1, 1∶1, 1∶2, 1∶3, 1∶4, 1∶5, and 1:10, respectively.In the indirect contact culture group, the supernatant of UC-MSCs was used as the conditioned medium to re-suspend BMECs.In the control groups, UC-MSCs and BMECs were cultured alone.The cell growth status in each group was observed at 0, 4, 8, 12, 24, 36, 48, 60, 72 h after culture, and cell proliferation was detected by cell counting kit-8 (CCK-8) assay.Results At 48 h, the optical density of the conditioned medium-BMECs group was significantly higher compared with the control groups (P<0.05).Meanwhile, the optical density in the direct contact group at a concentration ratio of 1∶2 reached the peak, which was extremely significantly higher compared with the control groups (P<0.01) and significantly higher compared with the other direct contact culture groups and the conditioned medium-BMECs group (P<0.05).Conclusions Co-culture of UC-MSCs and BMECs in serum-free culture medium is capable to promote the proliferation of BMECs, and the co-culture by cell-to-cell contact has a better effect.The optimal concentration ratio of UC-MSCs to BMECs is 1∶2, and the optimal culture time is 48 h.
