CRISPR-on system for the activation of the endogenous human INS gene.

Advances in the field of epigenetics have allowed the design of new therapeutic strategies to address complex diseases such as type 1 diabetes (T1D). Clustered regularly interspaced short palindromic repeats (CRISPR)-on is a novel and powerful RNA-guided transcriptional activator system that can turn on specific gene expression; however, it remains unclear whether this system can be widely used or whether its use will be restricted depending on cell types, methylation promoter statuses or the capacity to modulate chromatin state. Our results revealed that the CRISPR-on system fused with transcriptional activators (dCas9-VP160) activated endogenous human INS, which is a silenced gene with a fully methylated promoter. Similarly, we observed a synergistic effect on gene activation when multiple single guide RNAs were used, and the transcriptional activation was maintained until day 21. Regarding the epigenetic profile, the targeted promoter gene did not exhibit alteration in its methylation status but rather exhibited altered levels of H3K9ac following treatment. Importantly, we showed that dCas9-VP160 acts on patients' cells in vitro, particularly the fibroblasts of patients with T1D.

Effects of season and superovulatory treatment on embryo yields in fine-wool Merinos maintained under field conditions.

The aim of the study was to assess the effects of superovulatory treatment (multiple FSH-dose vs single-shot FSH treatment) and seasonality on embryo yields in fine-wool Merino ewes. Treatment based on multiple FSH-dose consisted of 200 mg of FSH (Folltropin(®)) administered in seven decreasing doses. Single-shot treatment consisted of a single dose of 70 mg of FSH + eCG. In ewes treated with multiple FSH doses, number of recovered embryos was higher (6.0 ± 0.5 vs 3.5 ± 1.0), while non-fertilization rate was lower (12.8 ± 3.9 vs 40.3 ± 9.5) during the breeding season when compared to the non-breeding season (p < 0.05); although similar values of recovered Grades 1-2 embryos were observed between seasons. During the breeding season, proportion of responding ewes (98.1 vs 57.1%), ovulation rate (13.9 ± 0.8 vs 3.2 ± 1.2), recovered structures (7.9 ± 0.6 vs 1.7 ± 0.7), total recovered embryos (6.0 ± 0.5 vs 1.2 ± 0.6) and good-quality embryos (5.1 ± 0.5 vs 0.9 ± 0.6) were higher for the multiple FSH-dose treatment than for the single-shot protocol. In a similar way, in the non-breeding season, ovulation rate (11.3 ± 1.8 vs 6.0 ± 1.1) and recovered structures (6.6 ± 1.2 vs 2.7 ± 0.6) were higher for the multiple FSH injections protocol than those for the single-shot treatment, resulting in higher recovered Grades 1-2 embryos (3.2 ± 0.9 vs 1.4 ± 0.5). Current results indicate that seasonal anestrus affected embryo yields when applying multiple FSH-dose superovulatory treatment in Merino ewes, by decreasing the number of recovered embryos although the number of recovered good-quality embryos was not affected. During both seasons, multiple FSH injections produced higher ovarian response and number of viable embryos than the single-shot treatment.

Chemical activation with a combination of ionomycin and dehydroleucodine for production of parthenogenetic, ICSI and cloned bovine embryos.

The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 µM Io for 4 min followed by 5 µM DhL concentration and after 3-h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6-dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI-embryos or SCNT-embryos is reported here for first time.

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species.

Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.

Preselection of high and low ovulatory responders in sheep multiple ovulation and embryo transfer programs.

The present study evaluated the feasibility of carrying out an easy-to-handle and cost-efficient test for the preselection of high- and low-ovulatory responder ewes under superovulatory protocols. The test was based on the assessment of the number of ovulations obtained in response to the administration of a single-shot eCG treatment. The predictive value of the test was determined by comparing the number of ovulations with yields obtained in response to a multiple-dose FSH treatment. In addition, the study determined possible effects of follicular status at first FSH dose and their relationship with subsequent ovarian response. A total of 31 Merino ewes received hormonal treatment comprising the administration of 800 IU of eCG at the end of progestative treatment. Twenty-three days later, multiple-dose FSH treatment (80-mg FSH, in six decreasing doses between Days 12 and 14 of a second progestative treatment) was applied to the same ewes. The study showed a significant relationship between the number of corpora lutea obtained in response to eCG treatment with respect to those obtained in response to FSH treatment (r = 0.791; P < 0.05), which resulted in 84% recurrence rate. The number of embryos was greater for high-responder in relation to low-responder ewes (7.2 ± 3.7 and 4.0 ± 3.9, respectively; P < 0.05), whereas rates of recovery and fertilization were similar between groups (P > 0.05). Hence, there was a tendency for a higher mean of grades 1 and 2 embryos in high-responder in relation to low-responder ewes (6.1 ± 3.8 and 3.7 ± 4.0, respectively; P < 0.1). No significant relationship was found between the number of corpora lutea in response to FSH treatment and the number of small and total follicles at first FSH dose (P > 0.05). However, a negative low relationship was found between the presence of large follicles and the ovulation rate in response to FSH treatment (r = -0.361; P < 0.05). In conclusion, the results show the feasibility of carrying out an easy-to-handle and cost-efficient procedure for the preselection of embryo donors. The procedure was based on high recurrence rate between hormonal treatments, which in turn accounts for a distinctive ewe ovulatory response.

Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors.

The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.

Procedure for maximizing oocyte harvest for in vitro embryo production in small ruminants.

Possible effects of repeated hormonal treatments and laparoscopic ovum pick-up (LOPU) on the efficiency of oocyte recovery rate and quality were determined in sheep and goats. In six adult Merino sheep and five Criolla goats, ovarian status was synchronized by a prostaglandin F(2 alpha) analogue and the insertion of an intravaginal sponge 48 h later. Follicle development was stimulated by a single dose of FSH (60 mg NIH-FSH-P1) plus a single dose of equine chorionic gonadotrophin (eCG; 300 UI). The first FSH/eCG doses were administered 48 h after the sponge insertion, being repeated every 4 days to complete a total of four treatments in sheep and three in goats. Follicles in both ovaries were categorized according to their diameter and follicular fluid was aspirated under laparoscopic observation without a vacuum pump. In sheep, during a 12-day-period, a total of 347 follicles were aspirated with a recovery rate of 46.9%. In goats, during an 8-day-period, 219 follicles were aspirated with a recovery rate of 45.6%. In both species, there were no significant differences in the number of aspirated follicles, oocyte recovery rate and good quality oocyte recovery rate. However, in sheep the oocyte recovery rate was higher for large follicles, whereas in goats no such effect was detected. In summary, current results indicate that retrieval of oocytes can be maximized, without affecting oocyte quality, by repeating 'oneshot' FSH/eCG regimes and LOPUs at intervals as short as 4 days.