Oviduct-specific expression of tissue plasminogen activator in laying hens

Abstract

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.

Generation of transgenic chicks using an oviduct-specific expression system

“…Kaleri et al plasmid was double-digested with XhoI and BamHI, the PCR product of tPA was subcloned and the resultant vector was named pL-tPAGFP plasmid. The ovalbumin PCR product was subcloned into pL-tPAGFP plasmid and the resultant vector was named pL-2.8OVtPAGFP (Kaleri et al, 2011).…”

Generation of transgenic chickens by the non-viral, cell-based method: effectiveness of some elements of this strategy

“…Among the non-viral systems for the delivery of a gene into PGC, electroporation (van de Lavoir et al 2006 ; Oishi 2010 ) and lipofection (Naito et al 1998 ; Furuta et al 2010 ; Kaleri et al 2011 ) techniques have been used to transfer DNA. The main parameters that affect electroporation effectiveness are: pulse amplitude, pulse duration, number of delivered pulses, osmotic pressure (Kotnik et al 2003 ).…”

Gene editing in birds takes flight

“…Most transgenic chickens bioreactor platforms use regulatory regions from albumen (egg white) expression-specific loci, such as ovalbumin (OVA) locus, with high levels of target protein secreted by cells in the magnum of the oviduct as a result (Zhu et al 2005 ; Lillico et al 2007 ). Recent reports have demonstrated the production of antimicrobial peptides (Liu et al 2015a ), monoclonal antibodies for breast cancer therapy (Oishi et al 2011 ), epitope peptides for pollen (Kawabe et al 2012 ) and tissue plasminogen activator for anti-thrombotic therapies (Kaleri et al 2011 ; Lee et al 2013 ) in egg white. In fact, the first egg-specific pharmaceutical protein, Kanuma, was recently approved for treatment of lysosomal acid lipase deficiency (Sheridan 2016 ).…”