Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Interspecies Reconstructed Embryonic Cell Interaction between Campbell Hamster (Phodopus campbelli) and Mice (Mus musculus)

Interspecies embryo transfer is a seldom-used method to increase the successful conservation of endangered species. The study aimed to determine the potential development of interspecies reconstructed embryos. The present study used two animal models, Campbell hamsters (Phodopus campbelli) and mice (Mus musculus). The isolated inner cell mass (ICM) of hamster embryos were injected into the mice embryos. The embryos were transferred to the pseudopregnant mice using non-surgery embryos transfer methods. The fetuses were collected at day 13.5 of gestation for morphometric measurement and cytochrome b (Cyt b) analysis which used to determine the species of obtained fetuses. The results showed that the viability, pregnancy rate, and embryonic implantation ability of the interpecies reconstructed embryos did not differ significantly (p>0.05) compared to non-reconstructed embryos. Morphometric measurement showed that the crown-rump (CR) and the weight of fetuses in the reconstructed group were significantly higher than non-reconstructed group (p<0.05). According to Cytb analysis, the species of obtained fetuses were mice, while the population of hamster cells were found only in the blighted ovum (resorption). Therefore, it can be concluded that interspecies reconstructed embryos are able to implant. However, the population of mice cells are only found to develop.

P–806 Neogametogenesis via oocyte replication

Study question Can full preimplantation embryo development be achieved from artificial oocytes created through nuclear transfer of a haploid pseudo-blastomere (HpB) into a recipient ooplast? Summary answer It is feasible to replicate the female genome and generate novel sibling oocytes that can yield full preimplantation embryo development, albeit at a reduced rate. What is known already A limitation of assisted reproduction is the number of available oocytes for embryo creation. It is feasible to utilize a somatic cell nucleus to construct novel oocytes through a process known as haploidization, in which a reverse meiosis occurs after SCNT. Similarly, producing haploid parthenogenetic constructs can generate HpBs, useful for genetic testing at the pre-fertilization level or for reproduction. It is feasible to use a HpB as a nuclear donor since it has already completed homologue segregation. Study design, size, duration This is prospective translational animal model study. Over 6 months, 556 oocytes were manipulated for the experimental group, and 158 control oocytes were employed. B6D2F1 HpBs were used to establish the procedure and acquire expertise. FVB HpBs were subsequently introduced for genetic variance. Experimental and control embryos were cultured in a time-lapse incubator (up to 96h). Cleavage parameters were compared to control. Two-sample T-tests and one-way ANOVA with Bonferroni correction were employed for statistical analysis. Participants/materials, setting, methods A cohort of oocytes was harvested from B6D2F1 or FVB superovulated mice and artificially activated by 8% ethanol. At the 8-cell stage, HpBs were exposed to nocodazole. Another cohort of B6D2F1 oocytes was enucleated for recipient ooplasts. HpBs were individually transferred into the perivitelline space of the ooplasts alongside inactivated Sendai virus. After fusion, reconstructed oocytes with spindle development were fertilized by piezo-actuated ICSI using B6D2F1 spermatozoa. Unmanipulated and fertilized B6D2F1 oocytes served as control. Main results and the role of chance A total of 158 control oocytes underwent ICSI with a 67.7% survival rate; of these, 65.4% developed to the blastocyst stage. For artificial oocyte activation (AOA), up to 10 oocytes were activated for each experiment, yielding 8 HpBs per activated oocyte. For the experimental group, 556 oocytes underwent enucleation with a 96.4% survival rate. Nuclear transfer of HpBs resulted in a 93.2% survival rate, consistent for those derived from BDF and FVB. Reconstructed oocytes showed appropriate development of a novel pseudo-meoitic spindle at a rate of 63.7% for B6D2F1 HpBs and 75.5% for FVB HpBs, and ICSI yielded a 67.1% and 57.7% survival rate, respectively. The fertilization rate for the reconstructed oocytes was 64%. Control oocytes underwent ICSI with a 67.7% survival rate. When evaluating time-lapse parameters, reconstructed embryos created via blastomere nuclear transfer showed asynchrony compared to controls beginning as early as the stage of pronuclear fading. While the majority of reconstructed embryos arrested at the 4-cell stage, of those that progressed, 11.3% of those using BDF HpBs and 14.6% of those using FVB HpBs developed to the fully expanded blastocyst stage. This corresponds to a total of 23 reconstructed embryos that developed to the morula or blastocyst stage. Limitations, reasons for caution While we used single-well embryoscope culture for morphokinetic data collection, group culture is superior to single-embryo culture for mice. Thus, developmental rates may be underestimated by this protocol. Implantation and successful pregnancy are also needed to support the clinical utility of this method in generating gametes. Wider implications of the findings: For women with diminished ovarian reserve, oocyte yield and age-related aneuploidy are limitations to achieving genotyped offspring. Nuclear transfer of HpB can generate sibling oocytes while maintaining genetic information. This model represents a promising path for expanding oocyte yield, allowing genetic assessment of sibling oocytes, and enhancing chances of procreation. Trial registration number none

15 Blastocysts altered CDX2 and SOX2 gene expression and pregnancy failure after embryo transfer in yak heterospecific somatic cell nuclear transfer

Heterospecific cloning is a tool for the genetic rescue of endangered animals. Our objective was to evaluate the effects of heterospecific yak (Bos grunniens) cloned embryo aggregation on the expression levels of NANOG, OCT4, CDX2, and SOX2 genes, and to compare with IVF, parthenogenetic zona-free (P-ZF), and homospecific bovine cloned embryos (BB1x). Oocytes were recovered from the ovaries of slaughtered cows and invitro matured for 22h. The zona pellucida was removed by protease treatment and then mature oocytes were enucleated by micromanipulation. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with a somatic donor cell followed by electrofusion (with two 30-µs pulses of 1.2 kV/cm, 0.1s apart). Two hours after fusion, reconstructed embryos were activated using ionomycin followed by 6-(dimethylamino)purine (6-DMAP) treatment for 3h and cultured in synthetic oviductal fluid (SOF) medium for 7 days. The experimental groups were IVF, P-ZF, BB1x, heterospecific yak-bovine cloned embryos (1 embryo per microwell, YB1x), and heterospecific yak-bovine cloned embryos aggregated (2 embryos per microwell, YB2x). In all experimental groups, cleavage and blastocyst rates were assessed 7 days after activation. In addition, 5 blastocysts were pooled for each biological replicate, and pluripotency-specific genes (NANOG, SOX2, CDX2, and OCT4) were analysed by quantitative PCR. Data were analysed by the ΔΔCT method using the geometric mean of ACTB (actin) and GAPDH as internal standard followed by one-way ANOVA. Cleavages rates were significantly lower in the YB1x group compared with the other groups. Moreover, blastocyst rates in YB2x (31.34%, n=67) were significantly higher than in YB1x (13.86%, n=101) and BB1x (13.33%, n=45) groups, but there were no significant differences compared with the IVF (43.82%, n=89) and P-ZF (25%, n=68) groups. In contrast, although no significant differences were observed among groups in the expression of NANOG and OCT4 genes, the expression of CDX2 was lower in YB2x and YB1x blastocysts compared with the BB1X, P-ZF, and IVF (control) groups. In addition, a decrease in SOX2 gene expression was observed in the YB2x and YB1x blastocysts compared with the BB1X group. Blastocysts from YB1x (n=5) and YB2x (n=18) groups were transferred to recipient cows (n=23) on Day 7. Forty days after embryo transfer, presence of uterine fluid was detected by ultrasound in 3 recipient cows (from YB2x), suggesting embryo loss. In concordance with our previous reports, yak heterospecific SCNT blastocysts showed underexpression of CDX2 and SOX2 compared with the overexpression observed for these genes in bovine homospecific SCNT blastocysts. Thus, yak heterospecific SCNT blastocysts may have compromised developmental competence associated with altered expression of CDX2 and SOX2 that cannot be rescued by the aggregation of 2 reconstructed embryos.

19 Success of handmade cloning in a commercial setting

The purpose of this field trial was to determine whether handmade cloning could be used in a commercial setting to produce, transport, and implant embryos into recipients and to determine blastocyst and pregnancy rates. Donor animals and recipients were housed on 2 commercial farms, Farm A and Farm B. Ear notches were collected, grown in DMEM 10% FCS and 1% penicillin-streptomycin, and incubated at 38°C. Ovaries from a local abattoir were processed to collect cumulus-oocyte complexes for cloning. The cloning process included the following: (1) addition of demecolcine to maturation media at 22h, (2) bisection at 24 to 26h, (3) fusion at 25 to 27h, (4) activation at 30h, and (5) culture at 36h. After maturation, the cumulus cells were removed from the oocytes by incubating in 0.1% (wt/vol) hyaluronidase in HEPES-buffered TCM-199 with 2% (vol/vol) steer serum (T2) for 5min, followed by vortexing for 3min. The resulting cumulus-free oocytes were incubated in maturation media containing 0.5µg mL−1 of demecolcine for 2h. Next, the zona pellucida was removed with 0.2% (wt/vol) pronase in T2. An ultrasharp cutting blade was used to bisect the oocytes under a stereomicroscope, producing karyoplasts containing extrusion cones and cytoplasts. Fusion of 2 cytoplasts with a fibroblast was performed on a BTX fusion slide (San Diego, CA, USA) using a single direct current pulse of 100V for 9 µs. After fusion, the reconstructed embryos (REC) were incubated in SOFaaci for 3h until activation. The REC were activated with 10µM calcium ionophore for 5min in T2, followed by incubation in SOFaaci containing 2mM DMAP for 6h. Activated REC were individually cultured in well-of-the-wells (Vajta et al. 2000Mol. Reprod. Dev. 55, 256-264) containing SOFaaci without serum in 6% CO2, 5% O2, and 89% N2 for 7 days. For transport, 2-mL transfer tubes were filled with 400 uL of SOFaaci; overlayed with oil; gassed with 6% CO2, 5% O2, and 89% N2; loaded with 1 embryo per tube; and placed into a 39°C portable incubator. On Farm A, 34 REC were produced, with 13 developing to blastocyst stage (38% blastocyst rate). After a 1.5-h transport, 7 grade 1 expanded blastocysts were implanted into 7 synchronized recipients. At the 90-day pregnancy check, 3/7 (42%) were pregnant. On Farm B, 35 REC were produced, with 14 grade 1 morulas or early blastocysts developing (40% blastocyst rate). After a 6-h transport, 9 morulas or early blastocysts were implanted into 9 synchronized recipients. At the 90-day pregnancy check, 2/9 (22%) were pregnant. Overall, 5/16 (31%) of recipients remained pregnant by month 8 of gestation. In conclusion, handmade cloning is a practicable method to produce, transport, and implant embryos into recipients in a commercial setting.

Zebularine significantly improves the preimplantation development of ovine somatic cell nuclear transfer embryos

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P&lt;0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5nM zebularine for 12h compared with the control group (25.4±1.6 vs 11.8±1.7%, P&lt;0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P&lt;0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

Impact of different sources of donor cells upon the nuclear transfer efficiency in Chinese indigenous Meishan pig

Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for both agricultural and biomedical research. However, its efficiency is crucially dependent on the source of nuclear donor cells. In this study, we compared the cloning efficiency by using three lines of donor cells that are derived from fetal, newborn and adult fibroblasts of Chinese indigenous Meishan pig. We showed that cleavage rate and blastocyst formation rate of the reconstructed embryos were not significantly different between the fetal (80.7% and 15.6%) and newborn ear skin (77.5% and 12.3%) fibroblast groups (p>0.05), but in both groups these indices were significantly higher than that found in the adult ear skin (70.5% and 8.8%; p<0.05). Reconstructed embryos derived from fetal, newborn, and adult ear skin fibroblasts were transferred to four surrogates, respectively. For the fetal, newborn, and adult ear skin fibroblasts, the number of pregnancies were two (50.0%), two (50.0%), and one (25.0%), respectively, and the number of deliveries were two (50.0%), one (25.0%), and zero (0.0%), respectively. Seven and two cloned piglets were obtained from the fetal and newborn ear skin fibroblasts respectively, while no piglets were obtained from the adult ear skin fibroblasts. Two cloned piglets from the newborn ear skin fibroblasts died shortly after birth because of neonatal asphyxia caused by dystocia. The birth weights of the piglets derived from the fetal and newborn ear skin fibroblasts were 1230.5 and 1310.0g, respectively, which were statistically insignificant (p>0.05), but both were significantly higher than that of the control groups (p<0.05). Microsatellite analyses demonstrated that the genotypes of all cloned piglets were identical to their donor cells. Therefore, cloned pigs were successfully produced using two sources of donor cells isolated from the fetal and newborn ear skin fibroblasts of Meishan piglet, and indicating a better cloning efficiency than that obtained from adult fibroblasts. We concluded that the nuclear donor cell lines have significant impact on the developmental competence of cloned embryos as well as on the cloning efficiency of Meishan pig.