Recently, we published on the efficient production of transgenic cattle using the DNA transposon system (Yum et al. 2016 Sci. Rep. 6, 27185). In that study, 8 transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon) via microinjection of zygotes. In the analysis of their genomic stability using next-generation sequencing, there was no significant difference in the number of genomic variants between transgenic and nontransgenic cattle. In this study, we have described current status of those transgenic cattle in term of health, germ-line transmission, and application. All the transgenic cattle have grown up to date (the oldest being 30 months old, the youngest being 12 months old) without any health issue. In general blood analysis, there were not any significant changes between transgenic cattle and wild type. Because the transgene (green fluorescent protein; GFP) expression is constitutively active and has strong expression, it could be visualised without fluorescence equipment. One of transgenic male cattle reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for IVF. In every IVF replication, around 80% blastocysts expressed the GFP. Over 36 GFP blastocysts were frozen for embryo transfer in the future, and we are planning to crossbreed for generating homozygotic transgenic cattle. Another application is to use the GFP locus to gene-edit the transgenic cattle, as long-term expression of transgene did not affect their health. In 1 cell stage, embryos produced using GFP frozen-thawed semen, single guide RNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of single guide RNA target site, were co-injected and RFP was observed. Knockout/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated that transgenic cattle via transposon are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the target region for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on disease resistance and food production. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).
Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Arachidonate that is esterified predominantly in phospholipids is a precursor of eicosanoids. After its release by the action of cytosolic phospholipase A2 (cPLA2), arachidonate can be converted to prostaglandins, thromboxanes, and leukotrenes via the cyclo-oxygenase (COX1 and COX2) and lioxygenase pathways, respectively, depending on the cell type. It is reported that eicosanoids have influence on inflammation, vascularization, and parturition. Then again, free arachidonate released from the plasma membrane is reesterified into phospholipids to prevent constant synthesis of potent eicosanoids. In the rodent, vasoactive prostaglandins are implicated in the implantation process. To further clarify ACS4 gene expression during pregnancy, we examined developmental expression in the peri-implantation uterus of the normal mouse and regulation of ACS4 by steroid hormone in ovariectomized mice treated with estradiol-17β (E2) and/or progesterone (P4). Adult female mice (BDF1, 6 weeks old) were mated with fertile males of the same strain. The morning on which a vaginal plug was found was designated Day 0.5 of pregnancy. Mice were sacrificed between 09:00 and 10:00 h on Days 2.5 to 6.5 of pregnancy. To induce and maintain delayed implantation, mice were ovariectomized on the morning (09:00 to 10:00 h) of Day 3.5 of pregnancy and received a daily injection of P4 from Days 4.5 to 6.5. To terminate the delay and induce implantation, the P4-primed delayed mice were given an injection of E2 on Day 6.5. COX1, COX2, cPLA2, and ACS4 mRNA were analyzed by Northern blot analysis and real-time PCR. The expression of COX1 mRNA did not show much variation on the various days in the normal pregnancy and ovariectomized mice. The levels of COX2 mRNA were high on Day 4.5 of pregnancy and dramatically decreased on Day 5.5 of pregnancy. In the ovariectomized mouse uterus, COX2 mRNA levels declined rapidly at Day 4.5 of pregnancy. The expression of cPLA2 mRNA was not altered in the normal pregnancy. However, the expression was increased in the ovariectomized mouse as compared with that in the normal pregnancy mice. An ACS4 transcript was already present in the uterus at low level on Day 3.5 until the initiation of attachment reaction after which the expression was up-regulated. In the ovariectomized mouse uterus, ACS4 mRNA was increased at Day 4.5 of pregnancy as compared with the normal pregnancy mice. To determine whether ovarian steroids influenced the induction of ACS4 gene, ovariectomized mice were treated with (E2) and/or (P4). Treatment with P4 maintained the expression of ACS4 mRNA in the ovariectomized mouse uterus. In contrast, combined treatment with P4 and E2 modestly decreased the levels of ACS4 mRNA as compared with Day 7.5 of normal pregnancy. Overall, these results suggest that the ACS4 gene is regulated in the implantation process and influenced by ovarian steroids.
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