ABSTRACT
The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.
ABSTRACT
Oral administration of specific egg yolk immunoglobulin (IgY) is effective against a number of gastrointestinal pathogens. However, the activity of orally administered IgY is reduced rapidly, since IgY is sensitive to pepsin and low pH. In this study, hydrogels containing acrylamide and acrylic acid were synthesized and used to encapsulate IgY. The capacity of these structures to load, protect and release IgY and the interaction between IgY and hydrogels by FTIR spectroscopy were studied. The particle size and swelling percentage of hydrogels were highly dependent on the pH of the buffer solution. As expected, pH-sensitive hydrogels had a high IgY loading percentage (99.2 ± 12.9 mg IgY/mg hydrogel) at pH 7.4. It means that each gel piece incorporated approximately 8.4 ± 1.1 mg IgY. The results showed that the hydrogels could efficiently incorporate IgY and retain it inside the polymer network at pH <2.2. However, IgY was slowly released at basic pH and a high percentage remained inside. The IR spectra show that IgY interacts with the hydrogel in its network with extended hydrogen bonds. The present study demonstrates that hydrogels particles can efficiently incorporate the IgY but cannot show a controlled and sustained release of IgY in simulated intestinal fluid probably due to hydrophobic interactions with the polymer network. The stability of IgY in simulated gastric fluid was greatly improved by encapsulation in hydrogels. This approach provides information about a novelty method for delivery of IgY for the prevention and control of enteric diseases.
ABSTRACT
Early pregnancy factor (EPF) is an immunosuppressant that promotes maternal immune system tolerance of the allogenic fetus. Little is known about localization of this factor in different tissues and nothing has been reported about localization in swine reproductive and placental tissues. We determined the concentration of EPF in serum of gilts and porcine placenta conditioned medium (PPCM). We also analyzed the expression of EPF in different reproductive tissues of pregnant gilts at 10, 30, 60 and 90 days of pregnancy. EPF concentration in serum and PPCM was determined by western blot and densitometry. EPF expression in reproductive tissue was assessed by immunohistochemistry. The highest concentration of EPF was observed at 30 days in serum and PPCM; the concentration was higher in PPCM than in serum at the stages we evaluated. All reproductive tissues from the gestational stages analyzed showed specific labeling of EPF, but this labeling did not appear in non-pregnant gilts. At 30 days pregnancy, the EPF expression in the ovary was predominantly in follicular lutein cells, probably owing to its function as a luteotrophic factor. In the oviduct, EPF was expressed in unciliated secretory epithelial cells and in the cilia of ciliated cells. In the placenta, EPF was expressed in the fetal portion (mesoderm chorioallantois and epithelium of endoderm). EPF acts as an autocrine and paracrine growth factor for the trophoblast during the peri-implantation period.
