6 PRODUCTION OF GONADOTROPIN-RELEASING HORMONE II RECEPTOR KNOCKDOWN SWINE

Abstract

The second mammalian isoform of GnRH (GnRH-II) is highly conserved from bony fish to humans. However, coding sequence for the receptor specific to this ligand contains reading errors in many species, suggesting the inability to produce a functional receptor. In contrast, the porcine GnRH-II receptor gene contains the appropriate sequence to produce functional protein. The objective of this study was to develop swine with reduced levels of endogenous GnRH-II receptors. Two potential target small hairpin RNA (shRNA1 and shRNA2) sequences specific to the porcine GnRH-II receptor were identified and subcloned into the lentiviral-based, pLVX-shRNA2 vector (Clontech) that provides both shRNA and fluorescent ZsGreen1 coexpression. Lentiviral particles were produced from each shRNA vector as well as a control vector using the Lenti-X HTX Packaging System (Clontech). The ability of shRNA1 and shRNA2 lentiviral particles to reduce porcine GnRH-II receptor mRNA levels was tested in a swine testis-derived (ST) cell line. The day before transduction, ST cells (2 × 10⁶) were plated in 100-mm plates containing high-glucose DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U mL^–1 of penicillin and 100 mg mL^–1 of streptomycin sulfate. Next, cells were transduced with 1.44 × 10⁷ viral particles per plate for 48 h. The cells were harvested, RNA was extracted and converted to cDNA and quantitative PCR was performed to determine relative levels of GnRH-II receptor mRNA. Values were normalized using the expression levels of 18s rRNA and compared with GnRH-II receptor mRNA levels in ST cells transduced with control lentiviral particles. Lentiviral particles containing either shRNA1 or shRNA2 sequences significantly reduced GnRH-II receptor mRNA levels (95 and 99%, respectively) compared with control particles (P < 0.05). Next, lentiviral particles containing the shRNA2 sequence (1.15 × 10⁹ infectious units mL^–1) were microinjected within the perivitelline space of in vivo-derived pronuclear zygotes (n = 15) using a Nikon diaphot inverted microscrope equipped with Eppendorf micromanipulators and an Eppendorf FemtoJet injection system. Microinjected zygotes were subsequently cultured in 50-μL drops of NCSU-23 under mineral oil in a humidified 5% CO₂ in air environment. Following 120 h of culture, 93% of the zygotes developed to the compact morula stage, whereas 73% formed blastocysts at 168 h. By 192 h, 80% of the microinjected embryos developed to the blastocyst or expanded blastocyst stages. Fluorescent microscopy revealed that all blastocysts expressed ZsGreen1, indicating a 100% transduction efficiency of shRNA2 lentiviral particles. Finally, embryos were surgically collected from white crossbred donor sows and transduced as described above. A total of 40, 33 and 23 microinjected zygotes were immediately transferred into 3 synchronized recipient females that will be allowed to gestate to term. The animals produced from this study will represent the first animal model to examine the physiological implications of reduced GnRH-II receptor levels.