Influence of Ovalbumin Gene Regulatory Elements on Tissue Specificity and Level of Transgene Expression

Abstract

A b s t r a c tThe use of lentiviral vectors for the genetic modification of embryonic chicken cells is regarded as one of the promising methods for producing transgenic poultry. In this case, it is very important to determine the regulatory elements of the ovalbumin gene, providing tissue-specificity and high transgene expression in cells of chicken oviduct. The aim of this work was to study the effect of the intron sequences and promoter of ovalbumin gene on tissue specificity and level of the transgene expression. For this purpose constructs based on lentiviral vector pWpxl, containing eGFP marker gene under control of the modified ovalbumin gene regulatory elements were obtained. Vector pW2.8 included a chromosomal DNA fragment of 2.8 kb comprising a first exon, intron sequence and part of the second exon of the ovalbumin gene; vector pW1.2 -chromosomal DNA fragment of 1.2 kb comprising a promoter and ovalbumin gene sequence (without the intron and the first exon) to the transcription initiation point; vectors pW131, pW225, pW315 -chromosomal DNA fragment, similar to the fragment of 1.2 kb in pW1.2 vector in which promoter (80 bp) was replaced by highly structured sequence of the birds -actin gene promoter region of 131, 225 or 315 bp respectively. The control vector pWCAGgfp included constitutive hybrid regulatory element comprising human cytomegalovirus early gene enhancer and birds' -actin gene promoter (CAG). Primary culture cells of chick oviduct and human fibrosarcoma cells 293T (control) were used as target cells for transfection. Viral preparation was added after a monolayer of cells reached concentration of 1-3½10 7 CFU/ml. eGFP expression was determined by fluorimetry in 72 hours after transfection. Low level of expression of eGFP gene controlled by chromosomal fragment of 2.8 kb leader region of the ovalbumin gene was confirmed in vitro using culture of chicken oviduct cells and 293T cell line: in vector pW2.8 recombinant protein expression level was up to 25 times lower compared to pWCAGgfp vector with a constitutive promoter CAG. Yet the eGFP expression levels for pW1.2 and pW2.8 constructs were identical, indicating the absence of introns' influence on the expression level of the recombinant DNA using this regulatory element. When the ovalbumin gene promoter was replaced by highly structured elements of -actin constitutive gene promoter the increase in the expression of eGFP in 2-3 times was observed, as well as the increased expression occurred with lengthening of the promoter region of -actin gene (vectors pW131, pW225, pW315). The achieved levels of expression with the use of exogenous -actin gene promoter were comparable with expression levels controlled by a constitutive promoter CAG, however when the promoter part of the ovalbumin gene was replaced by exogenous promoter (gene -actin), the deregulation of tissue-specific expression of eGFP was observed, indicating that transcription with tissue-specific ovalbumin promoter gene can be modulated or activated with exogenous enhancers.