Production Of Interferon-κ From Immortalized Primordial Germ Stem Cells Of Chicken

Transgenic technology can overcome the limitations of traditional animal breeding,accelerate the process of genetic improvement of germplasm,break down genetic barriers among species,and expand alternative genetic resources.It is one of the important factors affecting the future development of modern agricultural technology.Chickens have a short generation interval,easy feeding and management,relatively low cost,and high production performance.Stem cells(SCs)-mediated transgenic technology is currently considered to be one of the most effective transgenic approaches.Chicken primordial germ stem cells(PGCs)possess chicken genetic information and capability of stable germline transmission,transgenic chickens are expected to be obtained quickly and efficiently through genetic manipulation in vitro.However,the passage numbers of PGCs are limited,and these cells cannot be cultured in vitro for a long time,which limit their application in producing the transgenic animals.In this study,the in vitro isolation method and culture system of PGCs were optimized,and the immortalization of PGCs were realized by being transfected with human telomerase reverse transcriptase(hTERT)gene,which promoted their growth and proliferation,these immortalized PGCs also had stable pluripotent characteristics.After being transfected with PiggyBac(PB)Transposon carrier named IFN-κ-His-PiggyBac Dual promoter,the transgenic PGCs could express the recombinant protein factor Interferon-Kappa(IFN-κ),the isolated and purified IFN-κhad the ability to anti-newcastle disease virus(NDV).This establishment of system for producing IFN-κ using the immortalized PGCs is expected to provide new drugs for resisting viral diseases,and serve to basic research,animal husbandry production,human disease treatment and so on.1.Optimization of in vitro isolation and culture system for chicken PGCsThe efficiencies of isolating cells from chicken genital ridge with different combinations of digestive enzymes were compared by detecting cell count,cell death rate and colony formation rate,and the effects of cell feeder layer on the growth of PGCs were also investigated,followed by identifying the pluripotent characteristics of the generated PGCs,which aimed to optimize the in vitro isolation and culture system for chicken PGCs.The results showed that the total number of cells and the colony formation rate in 0.25%trypsin and 0.3%collagenase IV treatment group were significantly higher than these in 0.25%trypsin and 0.1%hyaluronidase treatment group(P<0.05),and the cell death rate in the former was significantly lower than that in 0.25%trypsin treatment group(P<0.01);The colony formation rate of PGCs in DF-1 feeder layer culture system was 7.13 ± 0.15%,which was significantly higher than that in NIH/3T3 feeder layer(4.92±0.4%),CEFs feeder layer(2.35±0.52%)and feeder-free system(0.48±0.04%)(P<0.01);The isolated PGCs were positive for alkaline phosphatase(AKP)staining,Periodic Acid-Schiff stain(PAS)staining and immunocytochemical staining for SSEA-1,Sox 2 and Oct 4,and expressed pluripotent stem cell marker genes including Nanog,Sox 2,GDF 3 and cPouV.They could also be differentiated into embryoid bodies(EBs),the expressions of marker genes of mesoderm(GATA 6),endoderm(AFP)and ectoderm(Sox 2)were identified in EBs.Therefore,these isolated PGCs show certain pluripotent characteristics.2.Immortalization of chicken PGCsPGCs were transfected with hTERT genes via eukaryotic expression vector namely pCI-neo-hTERT+EGFP for immortalization to enhance their abilities of proliferation and passage.The results showed that EGFP-positive cell colonies were screened from PGCs transfected with pCI-neo-hTERT+EGFP after being treated with G418;The plasmid transfection efficiency of PGCs on DF-1 feeder layer cells with the confluency rate of 80%was significantly higher than that of 40%(P<0.05)or 20%(P<0.01)DF-1 feeder layer;When the confluency rate of DF-1 feeder layer cells was 80%and the ratio of Lipofectamine LTX Reagent and plasmid DNA was 1:1,the transfection efficiency of PGCs was significantly higher than these in other treatment groups,respectively(P<0.05);EGFP+PGCs still retained the pluripotency characteristics of primary PGCs,and their growth curves indicated that their proliferation ability had been enhanced,and the expression of telomerase was significantly higher than that of primary PGCs(P<0.01).Therefore,the introduction of hTERT gene through the pCI-neo-hTERT+EGFP vector can enhance in vitro proliferation and passage number of PGCs,maintain their pluripotency characteristics,and induce their immortalization.3.Production of antiviral factor namely IFN-κ from immortalized PGCsThe immortalized PGCs were transfected with transposon vector namely IFN-κ-His-Dual promoter for expressing and generating IFN-κ.With the help of their connected His tag,IFN-κ had been purified through His nickel column,Western Blot was used to detect its expression,and its resistance to Newcastle disease virus(NDV)was determined by means of the pathological changes of CEFs and PGCs.The results showed that after the immortalized PGCs were transfected with the PB transposon vector for IFN-κ,PGCs carrying hTERT and IFN-κ genes were successfully obtained;Proteins were extracted from the lysed hTERT+IFN-κ+PGCs and purified by affinity chromatography of His nickel column,the concentration of the purified IFN-κ was greater than 500 μg/mL as shown by BCA concentration test,and the relative expression level of IFN-κ detected by Western Blot was 1.231;The anti-NDV activities of IFN-κ on CEFs and PGCs were respectively 640 U/mL and 320 U/mL using microcytopathic inhibition method and Reed-Muench algorithm.Therefore,IFN-κ with anti-NDV efficacy can be prepared by using immortalized PGCs and being transfected with PB transposon vector.Conclusions:0.25%trypsin and 0.3%collagenase Ⅳ are suggested to digest genital ridge of chicken embryo,the isolated cells should be cultured on the DF-1 cell feeder layer in vitro,with the aid of LIF,SCF,IGF-1,bFGF,chicken serum and fetal bovine serum,PGCs with characteristics of pluripotent stem cells can be more efficiently obtained.When the confluency rate DF-1 feeder layer cells is 80%and the ratio of Lipofectamine LTX Reagent to plasmid DNA is 1:1,the introduction of hTERT gene in PGCs via pCI-neo-hTERT vector can increase the expression of exogenous telomerase,enhance the proliferation and passage ability of PGCs,making them immortalization.hTERT+PGCs still maintain their pluripotent stem cell characteristics.IFN-κ gene can be transfected into hTERT+PGCs via PB transposon vector,which can induce these cells to produce IFN-κ with anti-NDV efficacy;The PGC immortalization and IFN-κ preparation system established in this study can provide the technical platform for the preparation of genetic chickens and the production of novel medicinal proteins.