Expression Of Ovalbumin-fusion Protein In Chicken Using CRISPR/Cas9 Technology

Chicken oviduct bioreactor is considered to be one of the most promising strategies for production of recombinant proteins,for use in biomedical reseach and clinic.Due to the unique reproductive physiological of poultry,research of poultry bioreactor lags far behind that of mammalian bioreactor.The key to solve this problem is to construct a suitable foreign gene expression cassette and insert it into the poultry genome mediated by using primordial germ cells for efficient expression and secretion.The CRISPR/Cas9 technology is an efficient gene editing tool being used in many research fields.In this study,we used CRISPR/Cas9 technology to target the last exon of the chicken ovalbumin gene and inserts expression cassettes of foreign gene into the genome of chicken primordial germ cells(PGCs),which allow the fusion with ovalbumin and espressed specificall in oviduct.The results are as follows:First,in oeder to select a suitable linker peptide for use in fusion protein,different peptide linkers of recombinant proteins were compared.Petide linker(GGGS)3,(EAAAK)3 and 2A were used to ligate enhanced green fluorescent protein(EGFP)and cloned to p EGFP-N1 vector to construct fusion protein expression vectors(pOVAL-G3-PEGFP-N1,pOVAL-E3-PEGFP-N1,pOVAL-2A-EGFP-N1),linker-free fusion protein expression vector was used as control.The expression vectors were transfected into 293 T cells.The expression of GFP was observed in all transfected cells 12 h after transfection and the intensity of EGFP in cells transfected with pOVAL-2A-EGFP-N1 and pOVAL-E3-EGFP-N1 was significantly stronger than others.Western blot results revealed that the expression of fusion protein was detected in the extracts from all the transfected cells.The expression level of the fusion protein in the cell extract from pOVALE3-EGFP-N1 was the highest,while with the lowest expression was seen in pOVAL-EGFP-N1 group.And as espected,EGFP from the pOVAL-2A-EGFPN1 group was expressed as monomers instead of fusion protein in other groups.Secondly,in order to fused the EGFP protein with edogenous ovalbumin and express specifically in chicken ovidut,doner vectors E3-EGFP-CMV-mCherry and G3-EGFP-CMV-mCherry were constructed.And CRISPR/Cas9 plus HMEJ strategy was utilized to insert the expression cassettes into the last intron of ovalbumin.And 2A-EGFP-CMV-mCherry doner plasmid was used as control.The single-cell PCR showned that the knock-in efficiency was 41.7% in chicken PGC cells.And sequencing analysis revealed that 10 out of the 11 clones carred correct expression cassette after targeted integration.Finally,we attempts to generate germline chimeric chicken by using PGCs to express the fusion proteins.We co-translated the CRISPR/Cas9 plasmid and the donor plasmid E3-EGFP-CMV-mCherry into primary chicken PGC cells.The single cell PCR results showed that the targeted integration efficiency was 10%.The positive cells were purified by flow sorting and tranplanted into stage 15 chicken embryos to produce chimeric chickens.Migration of PGCs was subsequently examined RFP-positive cells were observed in the gonads isolated on day 5 after transplantation.RFP cells were also observed in the testis of the chickens 14 days after birth,indicating that the germline chimeras were successfully generated.In summary,we compare different linkers for used in protein fused with ovalbumin and specifically expressed in chicken oviduct.And we also inserted the expression cassettes into the last exon by targedted integration using the CRISPR/Cas9 technology and generate germline chimeras mediated by PGCs transplantation.Results of the current study would help to establish an efficient bioreactor using chicken model to produce protein of pharmaceuticl importance.