Oviduct-specific Expression Of Human Neutrophil Defensin 4 In Transgenic Chickens

Antimicrobial peptides (AMPs) are a class of biologically active small peptides with broad-spectrum antimicrobial activity which are recognized as novel therapeutic agents for their special mechanism of action. Defensins, a major class of AMPs, can be divided into a defensins and β defensins according to the structure they have. HNP4, encoded by the DEFA4 gene, is a kind of a defensin. The mature HNP4 is a small peptide of 33 amino acid residues with a molecular weight of 3.7 kD. Compared with other a defensins (HNP1-HNP3), HNP4 displays more than 100-fold higher activity against Escherichia coli, and four-fold higher activity against both Streptococcus faecalis and Candida albicans, and HNP4 is also able to inhibit the HIV and HSV virus.Animal bioreactor is a very powerful technique that can be used to produce recombinant proteins. Chickens oviduct is one of the most important animal bioreactors. The advantage of oviduct bioreactor is that the exogenous protein expressed in vivo can be directly secreted into the egg white, which could reduce the harm caused by the exogenous gene. The component of egg white is simple, so the recombinant proteins can be easily purified in vitro. However, the unique features of the reproductive system of birds, and the fact that their embryonic development occurs in a shelled egg make it difficult to obtain transgenic birds. In recent years, lentivirus and PGCs are considered to be the effective tools for preparing transgenic chickens. These methods are only successfully reported in the developed countries because of the low germline frequency.In the present study, we used lentiviral vectors to generate transgenic chickens, with the aim of producing chickens with eggs containing the HNP4 protein. The ovalbumin promoter containing both the steroid-dependent regulatory element (SDRE) and the negative regulatory element (NRE) was cloned and verified in vitro. The nucleotides with coding sequences of the matured HNP4 gene were synthesized, and the signal peptide of chicken lysozyme was added into the 5’end. We constructed pWPXL-Ova-HNP4 and pWPXL-Ova-HNP4-His expression vectors and both vectors were packaged into lentivirus. Genetically manipulated White Leghorn chickens were generated by injecting lentivral vectors into developing embryos (stage X). Out of the 669 injected eggs,218 chickens were successfully hatched and the hatch rate was 32.6%. Ten roosters whose semen were identified as positive for transgene were mated with wild type hens to produce G1 chicken. Fifteen G1 positive individuals from 1274 offsprings were obtained with frequencies ranging from 0.6% to 3.4%. The Southern blotting and Genome Walking results indicated that a single copy of the HNP4 gene was integrated into chromosomes 1,2,3,4,6 and 24 of the chickens. RT-PCR and immunofluorescence analysis of G1 and G2 chickens indicated that the HNP4 expression was restricted in oviduct tissue as expected. The level of HNP4 protein detected in the eggs white of G1 and G2 heterozygous chicken ranged from 1.65 μg/mL to 10.18 μg/mL and the expression varied greatly at both the transcriptional and translational levels among different insertion sites.In summary, we successfully produced transgenic chicken expressing HNP4 protein in egg whites and our results indicated that the HNP4 protein could be expressed stably from G1 to G2 chickens, which might provide a new clue for producing pharmaceutical proteins by using oviduct bioreactor.