Single step production of Cas9 mRNA for zygote injection.

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

The effect of leptin on luteal angiogenic factors during the luteal phase of the estrous cycle in goats.

Fibroblast growth factor 2 (FGF2), angiopoietin 1 (Ang1), and vascular endothelial growth factor (VEGF) are angiogenic factors implicated in the vascular development of the corpus luteum (CL). Each factor is regulated or influenced by leptin in non-ovarian tissues. Moreover, leptin and its receptor, ObRb, have been identified in luteal tissue throughout the luteal phase. Therefore, leptin is hypothesized to influence luteal vasculature through the regulation of FGF2, Ang1, and VEGF. Multiparous, cycling crossbred female goats (does) were allocated to early (n=12), mid (n=8), and late (n=11) stages of the luteal phase for CL collection. Luteal tissue was harvested and either snap frozen in liquid N2, paraffin embedded, or cultured with leptin (0, 10(-12), 10(-11), 10(-10), 10(-9), 10(-8)M). Tissue was analyzed for FGF2, Ang1, VEGF, ObRb, and leptin expression. Angiopoietin 1, FGF2, VEGF expression was higher (P≤0.001) in the mid-luteal stage than the early stage. Expression decreased (P≤0.001) during the late luteal stage with the exception of VEGF, which remained elevated. In contrast, leptin and ObRb were lowest (P≤0.003) during the mid-luteal stage compared to the early and late stages. All factors were detected in and/or around vessels in early stage tissue compared to mid and late stages. Leptin stimulated (P≤0.02) Ang1, FGF2, and VEGF expression only in early stage luteal cultures. Collectively, these data provide evidence that leptin may be involved in the luteal angiogenic process during the early stage of CL formation.