Efficient Source of Cells in Proximal Oviduct for Testing Non-Viral Expression Constructs in the Chicken Bioreactor Model and for Other <I>in Vitro</I> Studies

Abstract

Efficient source of cells in proximal oviduct for testing non-viral expression constructs in the chicken bioreactor model and for other in vitro studies. Folia Biologica (Kraków) 64: 37-46. This work shows the usefulness of chicken oviduct epithelial cells (COEC) in evaluating the efficacy of non-viral expression vectors carrying human therapeutic genes. Secondly, an efficient source of progenitor COEC for in vitro studies is described. Within the distal part of the oviduct, weak to moderate expression of a trans membrane glycoprotein (CD44) was observed. Single cells presenting only weak expression of CD44 were found in magnum sections. in vitro cultured oviduct cells originating from the distal oviduct were suitable for subculturing and showed a stable proliferation potential up to the 2nd passage. However, the pavimentous epithelial-like morphology of COEC was progressively lost over time and mainly a fibroblast-like monolayer was established in consecutive passages. Moreover, various commercial transfection agents including FuGENE6 and XtremeGENE9 DNA were used to optimize delivery of human interferon alfa-2a, (IFNá2a) a therapeutic protein gene under an ovalbumin promoter. The transfection efficiency of adherent COEC was estimated for up to 40% at a ratio of 6:1 of transfectant to pOVA5EIFN + GFP plasmid. Expression of IFNá2a was confirmed by western blotting in transformed COEC. In conclusion, the population of epithelial progenitor cells sourced from the distal oviduct can significantly contribute to in vitro culture of COEC, representing an efficient model to develop the production of avian bioreactors and other in vitro studies related to oviduct tissue.

Molecular signatures of epithelial oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica)

“…Strong induction of keratin genes is related with this function of the infundibulum. Previously, we have detected cytokeratins in chicken infundibulum by using immunohistochemistry technique, both in tissue and in vitro [ 6 ], which is in line with the results of the current study.…”

“…CD44-positive cell population showed the capacity for clonal growth and differentiation into tubal epithelial cells, particularly in the distal region of the tube [ 15 , 27 ]. We earlier showed a high immunochemical stain of CD44 in the distal oviduct of a hen [ 6 ]. SOX9 is a transcription factor in early epithelial lineage [ 28 , 29 ].…”

“…Both hen and quail oviduct cells secrete human therapeutic proteins after genetic modification [ 3 ]. Therefore the oviduct epithelium is a useful and fast in vitro model to test for the efficiency of viral [ 5 ] and nonviral genetic constructs [ 6 ] to study the modified secretome. Both quail and hen produce cellular substrates for the development of vaccines [ 7 ].…”

Secreting oviduct epithelial cells of Coturnix coturnix japonica (QOEC) and changes to their proteome after nonviral transfection

“…We recently reviewed avian cell‐based methods for the nonviral delivery of human exogenes and discussed the problematic aspects along with further recommendations . The most popular and studied nonviral transfection techniques for avian cells are the following: electroporation to cell membrane (Chojnacka‐Puchta et al) or to a nucleus membrane (nucleofection), lipofection or mediation using nonliposomal transfection agents . Electroporated cells are exposed to numerous side effects including Joule heating from electric current running through the electroporation reaction and nonhomogenous distribution of the electric field .…”

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

“…One promising model developed by our group (Kasperczyk et al 2012 ) could be the culture of chicken oviduct epithelial cells (COEC) that can be easily transfected with constructs based on oviduct-specific regulatory sequences, for example those derived from the ovalbumin or lysozyme genes. This unique, in-house-developed in vitro culture system for COEC has the potential to produce therapeutic proteins in vitro which would allow the verification of their functions and activity and, most importantly, of the efficiency of recombinant gene expression in the tubular gland cells of the oviduct (Stadnicka et al 2016 ).…”