piggyBac Transposition and the Expression of Human Cystatin C in Transgenic Chickens

Abstract

A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow's milk and hen's eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.

Keywords: bioreactor; human cystatin C; piggyBac transposon; transgenic chickens.

Figure 1

The piggyBac transposon-based vector and expression of human cystatin C (hCST3). (A) Schematic of the expression vector and chicken codon-optimized hCST3 with a His-tag. The CMV promoter controlled hCST3 expression and a puromycin-resistance gene was used as a transgene selection marker. The arrows indicate the primer sites in the hCST3 expression vector (hCST3, human cystatin C; PBTR, piggyBac terminal repeat; CMV, cytomegalovirus; EF1, elongation factor 1). (B) Detection of hCST3 expression in transfected, puromycin-selected DF1 cell lines. (C) Detecting the hCST3 transgene and its expression in transfected, puromycin-selected chicken primordial germ cells (PGCs).

Figure 2

Production of hCST3 transgenic chickens. (A) Detection of GFP-expressing hCST3-transgenic chicken PGCs. GFP-positive chicken PGCs were detected in the recipient embryonic gonads (6-day-old) after transplantation. (B) Production of hCST3 transgenic chickens through testcross analyses. Ultimately, 13 hatched chicks were identified as transgenic chickens by genomic DNA PCR analyses. ID# is the individual number of donor PGC-derived chicks.

Figure 3

Comparative histochemistry analyses of hCST3 transgenic chickens. There were no differences in the histochemical structure of the liver, muscle, or kidney between control and hCST3 transgenic chickens. Paraffin sections were stained with hematoxylin and eosin (magnification 20×).

Figure 4

Quantification and antimicrobial activity test of hCST3 in transgenic chickens. (A) Quantification of hCST3 in the egg white (EW) of transgenic hens by ELISA. hCST3 was detected in all of the transgenic hen eggs. (B) hCST3 was detected in the muscle of hCST3 transgenic chickens by Western blotting. (C) His-tagged hCST3 in muscle and egg white of hCST3 transgenic chickens was purified and detected by Western blotting after purification with Ni-NTA magnetic nanobeads. (D) Biofunctional activity of hCST3 from transgenic hen eggs. The purified hCST3 from transgenic hen egg white was transferred to Whatman paper disks at different concentrations: (1) control (ddH₂O), (2) elution buffer, and (3) 10, (4) 25, (5) 50, (6) 75, and (7) 100 ng hCST3/disc. Original western blot figures in Figure S1.