Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

Abstract

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyleneimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-N1 was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the beta-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

Fig. 1

Strategy for construction of recombinant vector

Fig. 2

Primary culture of chicken oviduct epithelium, day 5 (10×40)

Fig. 3

The EGFP gene expression in the plasma of cells (a) Oviduct epithelial cells; (b) Oviduct fibroblasts cells

Fig. 3

The EGFP gene expression in the plasma of cells (a) Oviduct epithelial cells; (b) Oviduct fibroblasts cells

Fig. 4

RT-PCR detection of laying hens tissues. The hens were injected with pOVLacZ plasmid and pOV plasmid Lane 1, negative control; Lanes 2–5, the heart, liver, spleen, oviduct of control B; Lanes 6–9, the heart, liver, spleen, oviduct of experiment group; Lane M, λDNA/Hind III+EcoRI Marker

Fig. 5

β-galactosidase activity in different tissues of laying hens in control B and experiment group

Fig. 6

β-galactosidase activity in eggs of laying hens in control B and experiment group