217 PRODUCTION OF A GONADOTROPIN-RELEASING HORMONE II RECEPTOR KNOCKDOWN SWINE LINE

Abstract

Unlike the native form of gonadotropin-releasing hormone (GnRH-I), the second isoform of GnRH (GnRH-II) is highly conserved throughout evolution and is ubiquitously expressed. The pig represents one of the few species possessing coding sequence for a functional receptor specific to GnRH-II (GnRHR-II). Binding of GnRH-II to its receptor has been linked to regulation of cell proliferation, feed intake, and the interaction between energy balance and reproductive behaviour. The objective of this study was to develop a porcine model with reduced levels of endogenous GnRHR-II to examine the biological role of this G-protein coupled receptor. Previously, we produced lentiviral particles from a vector overexpressing both small hairpin RNA (shRNA) sequence specific to the porcine GnRHR-II and cDNA encoding the fluorescent ZsGreen1 protein (pLVX-shRNA2; Clontech). Transduction of swine testis cells with these particles (1.44 × 10⁷ viral particles) reduced porcine GnRHR-II mRNA levels by 99% compared with control particles (P < 0.05). In the current study, pronuclear zygotes (n = 50) surgically collected from 1 white crossbred donor sow were microinjected into the perivitelline space with lentiviral particles containing the shRNA2 sequence (3.3 × 10⁸ IU mL^–1) using a Nikon diaphot inverted microscrope equipped with Eppendorf micromanipulators and FemtoJet injection system. A total of 40 microinjected zygotes were immediately transferred into the oviduct of 1 synchronized recipient female, resulting in the production of 5 healthy, live piglets (20% efficiency rate). Interestingly, 1 female exhibited green fluorescence, indicative of successful transgene integration and expression. Transgene integration was confirmed via conventional PCR using primers designed to amplify portions of the human U6 promoter driving the shRNA, the CMV promoter driving ZsGreen1 expression, and the multiple cloning site for incorporation of the shRNA sequence. Next, inverse PCR was performed to determine the location and number of integration sites. Sequencing analysis of PCR products revealed that a single integration site was present on chromosome 14, aligning with clone NW_003612067.1 with 99% identity and matching identities 448,946–448,37. The GnRHR-II knockdown (KD) female along with 2 female littermates were maintained and monitored during development. Attainment of puberty occurred at 149 days for the transgenic female and 145 and 151 days for littermate control gilts (P > 0.05). Upon exhibition of their third behavioural oestrus, females were bred and allowed to gestate to term. Litter size was similar between the GnRHR-II KD female (15 live piglets) and control littermates (15 and 16 live piglets). Of the 15 piglets produced, 5 (3 males and 2 females) were positive for green fluorescence, confirming germline transmission of the transgene and further evidence for a single integration site. The swine produced from this study represent the first animal model to examine the physiological implications of reduced GnRH-II receptor levels.