The Generation Of Transgenic Chickens Oviduct-specific Expressing Recombinant Human Lysozyme By Lentivirus

A new laid fertilized egg is a chick embryo which has already contained about60,000cells because of the unique feature of chicken reproductive system. The avian egg, with its solid sealed eggshell and large fragile yolk, is not as transparent as mammalian ova, making it a challenge to view for injection and manipulation. In recent decades, with the widely application of the lentivirus and the successful culturing of chicken primordial germ cells (PGCs), main efforts for manipulating the avian genome using viral vectors and PGCs, resulting in an exogenous gene transmitting rate as high as52.2%. Several recombinant proteins have been reported successfully expressed in the egg which, including bacterial β-lactamase, human interferon a-2b, human monoclonal antibody and single-chain Fv-Fc fusion protein. Here we report successful expression of recombinant human lysozyme (rhLY) in the egg white using the lentivirus as an exogenous gene deliverer.Lysozymes (LYs), which are known as muramidases/N-acetylmuramide glycanhydrolases, are found widely in milk, saliva, and tears in mammals and also in avian egg white. Chicken egg lysozyme (cLY) is composed of129amino acid residues and has a molecular weight of14.3kDa, whereas human lysozyme (hLY) is composed of130amino acid residues and has a molecular weight of14.7kDa. The cLY protein shares59%identity in acids with hLY protein. Despite the similarities between these two LYs with respect to number of amino acids and molecular weight, the anti-bacterial activity of hLY is approximately threefold greater than the anti-bacterial activity of cLY. Human lysozyme has the function of anti-bacterial, anti-viral and anti-tumor, and is completely nontoxic to human body, therefore often be used as a safe anti-infective material, natural preservative and food additive for infant food and beverages. However, natural human lysozyme can only be extracted from milk, urine, neutrophils and placenta resulting in a very low yield. Chicken oviduct bioreactor is expected to be a low-cost, large-scale way of production of human lysozyme. Meanwhile, the antibacterial properties of egg white can be attributed to the presence of lysozyme, thus, the addition of hLY to eggs should produce more stronger bacteriostatic effects and could therefore potentially prolong the shelf life of edible eggs and the storage period of hatching eggs. It has large potential economic benefits for the egg production and chicken breeding.In this study, we constructed a plasmid based on a replication-defective lentivirus construct containing a synthetic cLY signal peptide and a447bp synthetic human lysozyme cDNA sequence driven by a2.8-kb oviduct-specific ovalbumin promoter. By microinjecting the viral vector into the sub-germinal cavity of stage X chick embryos, chimeric transgenic birds could be produced with germline transgene copy numbers of approximately0.001-0.1. The incorporation of the human transgene into these birds was confirmed by PCR and Q-PCR analyses of semen DNA from Go males. Six Go semen-hLY-positive males were chosen for crossing with wild-type hens. A total of830chicks were screened by PCR, and five (0.60%) transgenic G1offsprings were identified. The only female of Gi transgenic chickens expressed recombinant human lysozyme (rhLY) at57.66±4.10μg/mL in egg whites and the G2transgenic hens sired by the Gi transgenic cock A011expressed rhLY at48.72±1.54μg/mL. The transgene has been demonstrated to be able transmitted through germline and expressed in the oviducts of transgenic hens by indirect immunofluorescence assay of oviductal sections and ELISA assay for rhLY in egg whites from the transgenic hens. Data of the RT-PCR and immunofluorescence analysis of G2hens indicated that the expression of the rhLY in terms of tissue-restricted expression.This research demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems for the production of therapeutic proteins and other applications.