INTRODUCTION

In recent decades, the global demand for therapeutic proteins has considerably increased. Although there are various platforms for the production of recombinant proteins, the avian production system, due to its significant advantages over other systems, has been taken into more consideration (Demain & Vaishnav, 2009; Houdebine, 2018; Ivarie, 2003, 2006; Maksimenko, Deykin, Khodarovich, & Georgiev, 2013; Raju, Briggs, Borge, & Jones, 2000). Recombinant human proteins produced in chicken cells in comparison to the produced proteins in plant, bacterial, or non-human mammalian cells, contain posttranslational modifications (including glycosylation) that are much more similar to those in proteins produced by human cells (Zhu et al., 2005). The proper posttranslational modifications are essential for successful therapeutic efficacy, prevention of unintended immune response, the long half-life of proteins, and biological activity in vivo (Elliott et al., 2004; Kodama et al., 2008; Rapp, Harvey, Speksnijder, Hu, & Ivarie, 2003).
Oligosaccharides on IgG in human and chicken are similar and comprise mainly N-acetylneuraminic acid (NANA), whereas other species contain N-glycolylneuraminic acid (NGNA) and NANA (e.g. rabbit), or only NGNA (e.g. sheep, goats, and cow) (Simon G. Lillico, McGrew, Sherman, & Sang, 2005; Raju et al., 2000; T. Shantha Raju, 2003). Therefore, avian expression systems represent desirable platforms for the production of recombinant human proteins.
In the early attempts to produce foreign proteins in avian systems, viral vectors containing a constitutive promoter (such as CVM promoter) were used to drive the expression. The use of these constitutive strong promoters had several disadvantages including variations in the level of protein expression, improper folding of the protein product, the possibility of promoter silencing, and toxicity due to their expression in a wide range of tissues. Thus, there has been an increasing trend toward the use of regulated promoters. Among these, the native hormone-dependent promoters have demonstrated to be efficient in transgene expression. For example, the ovalbumin promoter with its regulatory sequences has been used in the cultured primary oviduct cells or in the transgenic chickens for the production of exogenous proteins (Byun et al., 2011; Cao et al., 2015; Herron et al., 2018; Kodama et al., 2012; M. S. Kwon et al., 2018; S. C. Kwon et al., 2010; S G Lillico et al., 2007; T. Liu et al., 2015; Oishi, Yoshii, Miyahara, & Tagami, 2018; T. S. Park et al., 2015; Zhu et al., 2005). Despite the successful achievements and progress in this field, primary oviduct cell culture is difficult to perform and needs experimental requirements including efficient transfection of the primary cells, cell-cell and cell-substratum interactions, and dependency to steroid hormones (Yoshimura & Oka, 1990). Furthermore, the generation of transgenic chicken is elaborate, time-consuming, and requires highly skilled personnel for embryo-manipulation.
Hence, using a hormone-independent promoter to drive the transgene expression in a chicken cell line might be an easy and quick alternative method of production platform for human proteins. This system would allow us to use the significant advantages of production in chicken cells.
The regulatory sequences of the ovalbumin promoter, which has been extensively used in the chicken production systems are well-characterized (Dougherty, Park, & Sanders, 2009; Dougherty & Sanders, 2005; Haecker, Muramatsu, Sensenbaugh, & Sanders, 1995; Kato et al., 1992; Kaye, Bellard, Dretzen, Bellard, & Chambon, 1984; Kaye et al., 1986; Monroe & Sanders, 2000; H. M. Park, Haecker, Hagen, & Sanders, 2000; Sanders & McKnight, 1988; Schimke, McKnight, Shapiro, Sullivan, & Palacios, 1975; Schweers, Frank, Weigel, & Sanders, 1990; Sensenbaugh & Sanders, 1999; Wang et al., 1989). Although, there are many reports on the cis-acting regulatory sequences responsible for ovalbumin gene expression, other factors including trans-acting regulatory elements, nucleosomal rearrangements, histone modifications, the chromosomal structure of the gene locus, and the three-dimensional (3D) nuclear organization may play critical roles for its proper expression in oviduct cells (Bellard, Dretzen, Bellard, Oudet, & Chambon, 1982). Previous plasmid-based studies have shown that the deletion of the steroid-dependent regulatory element (SDRE; −900 to −732) and negative regulatory element (NRE; −308 to −88), as well as the linker between them in the ovalbumin promoter, increases the reporter gene activity driven by that (Haecker et al., 1995; Sanders & McKnight, 1988; Sensenbaugh & Sanders, 1999). These reports demonstrated that the presence of ovalbumin proximal promoter (−87 to +9) is sufficient for an estroid-independent expression. This led us to hypothesize that thein situ deletions of SDRE and NRE elements in the genome of a chicken non-oviduct cell line may also lead to the steroid-independent ovalbumin gene upregulation, while the trans-acting regulatory elements are still able to exert their effects.
In this study, we have deleted the ovalbumin distal promoter including SDRE and NRE elements in the genome of the DF1 fibroblast cell line via CRISPR/Cas9 system, and have analyzed the increased expression of theOvalbumin gene and the induced activity of an inserted transgene.