A novel tissue specific alternative splicing variant mitigates phenotypes in Ets2 frame-shift mutant models

E26 avian leukemia oncogene 2, 3′ domain (Ets2) has been implicated in various biological processes. An Ets2 mutant model (Ets2db1/db1), which lacks the DNA-binding domain, was previously reported to exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair and curly whiskers. Here, we generated new Ets2 mutant models with a frame-shift mutation in exon 8 using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as embryonic development stopped before E8.5, as previously reported. When we rescued them by tetraploid complementation, these mice did not exhibit wavy hair or curly whisker phenotypes. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 Ets2em1/em1 embryos. This exon 8-skipped Ets2 mRNA was translated into protein, suggesting that this Ets2 mutant protein complemented the Ets2 function in the skin. Our data implies that novel splicing variants incidentally generated after genome editing may complicate the phenotypic analysis but may also give insight into the new mechanisms related to biological gene functions.

Ets2 frame-shift mutant models express in-frame mRNA by exon skipping that complements Ets2 function in the skin

The Ets2 transcription factor has been implicated in various biological processes. An Ets2 mutant model, which lacks the DNA-binding domain (ETS domain), was previously reported to exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair.Here, we generated new Ets2 mutant models with deletions in exon 8 and with frame-shift mutations using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as previously reported. After rescuing with tetraploid complementation, homozygous mutant mice were generated, but these mice did not exhibit wavy hair phenotype. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 embryos. As this in-frame mutation contained the ETS domain, the exon 8-skipped Ets2 mRNA was likely translated into protein in the skin that complemented the Ets2 function. Thus, these Ets2 mutant models, depending on the cell types, exhibited novel phenotypes due to exon skipping and are expected to be useful in several fields of research.Summary statementNew Ets2 mutant models showed embryonic lethal phenotype by a placental abnormality but did not exhibit a wavy hair phenotype as a previous model.

Effect of exogenous transcription factors integration sites on safety and pluripotency of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs), generated from somatic cells, not only possess similar characteristics with embryonic stem cells (ESCs), but also present more advantages than ESCs in medical applications. The classical induction method that utilizes the integration of exogenous genes into chromosomes may raise the potential risk of the safety of iPSCs. To investigate the potential correlation between the integration sites of exogenous transcription factors (TFs) and iPSCs’ pluripotency and safety, the integration of exogenous genes in three iPSC lines, which met the golden standard of murine developmental assay (tetraploid complementation), were analyzed. Twenty-two integration sites of exogenous TFs were identified by nested inverse polymerase chain reaction (iPCR) and 39 flanking genes’ functions were analyzed by gene ontology (GO). In the 22 integrated sites, 17 (77.3%) were located in the intergenic regions and the remainder were located in introns far from the transcription start sites. Microarray analysis of the flanking genes in these cells showed that there was no distinct difference in expression levels between the iPSCs, ESCs and mouse embryonic fibroblast (MEF), suggesting that the integration of exogenous TFs has no significant influence on the expression of flanking genes. Gene ontology analysis showed that although most of the flanking genes were housekeeping genes, which were necessary for basic life activity, none of these 39 flanking genes have correlation with tumorigenesis or embryogenesis, suggesting that the integration sites hold low risk of tumorigenesis.

Generation of clonal male and female mice through CRISPR/Cas9-mediated Y chromosome deletion in embryonic stem cells

Mice derived entirely from embryonic stem (ES) cells can be generated in one step through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female all-ES mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR/Cas9-mediated deletion of the Y chromosome allowing generation of clonal male and female mice by tetraploid complementation. The monosomic XO female mice are viable and are able to produce normal male and female offspring. Direct generation of clonal male and female mice from the same mutant ES cells significantly accelerates the production of complex genetically modified mouse models by circumventing multiple rounds of outbreeding.

24 Asynchronic tetraploid complementation and embryo quality in domestic cat and Leopardus geoffroyi hybrid embryos

Fusion of 2-cell embryos generates tetraploid (4n) blastomeres with an increased commitment to trophectoderm. Complementation of embryos from endangered species with 4n blastomeres derived from a phylogenetically related domestic species could improve healthy pregnancy establishment after embryo transfer in domestic females. However, generation, development, and quality of tetraploid complemented embryos in felids remain unstudied. Therefore, our objectives were (1) to evaluate tetraploidy of 2-cell fused embryos; (2) to analyse the blastocyst cell number, distribution after synchronic (S) or asynchronic (AS) complementation, OCT4+ cells, DNA-fragmentation levels and CDX2 gene expression of IVF complemented embryos; and (3) to evaluate the developmental rates of tetraploid complemented Felis catus-Leopardus geoffroyi hybrid embryos. After ovariectomy, Felis catus oocytes were IVM and subjected to IVF. For Experiment 1 (n=66), 2-cell embryos (2n) were exposed to two 30-ms DC pulses at 8 kV cm−1 electric field in fusion media. Fused (4n) and nonfused embryos were cultured invitro in 50-μL drops of modified Tyrode's medium. Karyotype analysis was performed at Day 4. For Experiment 2 (n=24), zona-free IVF embryos were aggregated S (4-cell-2n/4-cell-2n) or AS (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n). For Experiment 3 (n=36), blastocysts generated by AS complementation (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n) were either fixed with 4% paraformaldehyde for immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick end labeling assay or saved in RNA-Later for RT-qPCR analysis. For this experiment, nonaggregated 2n and 4n blastocysts were used as a control. For Experiment 4 (n=60), IVM oocytes were co-incubated with Leopardus geoffroyi and Felis catus (control) spermatozoa and then 4-cell-2n heterologous embryos were complemented with 1-cell-4n homologous IVF embryos. Data were analysed by Fisher's exact test. Our results showed that 67% of the 2-cell fused embryos were 4n. Moreover, 82% of nonfused embryos were aneuploids compared with 78% of 2n embryos in the control group. The AS complemented blastocysts (4-cell-2n/1-cell-4n and 4-cell-2n/2-cell-2n) had significantly higher cell number compared with S complemented (4-cell-2n/4-cell-2n) or noncomplemented embryos. The AS complementation also increased the number of OCT4+ cells independently of the ploidy of the embryos. Interestingly, AS tetraploid complemented embryos had significantly lower number of cells with fragmented DNA. No differences were found in CDX2 expression among complemented embryos; however, noncomplemented 2n blastocysts showed a significantly lower expression compared with the others group. Finally, we observed that AS complementation of 2n hybrid embryos with 4n homologous embryos reached similar blastocyst rates, 70 and 88%, respectively. Our findings support the use of 2-cell fused embryos to generate 4n blastomeres and demonstrated that tetraploid complementation generates good quality embryos, providing evidences that encourage the use of this technology to improve the developmental competence of interspecific embryos after transfer.

A novel microRNA-based strategy to expand the differentiation potency of stem cells

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

Cables2 Is a Novel Smad2-Regulatory Factor Essential for Early Embryonic Development in Mice

ABSTRACTCDK5 and Abl enzyme substrate 2 (Cables2), a member of the Cables family that has a C-terminal cyclin box-like domain, is widely expressed in adult mouse tissues. However, the physiological role of Cables2 in vivo is unknown. We show here that Cables2-deficiency causes post-gastrulation embryonic lethality in mice. The mutant embryos progress to gastrulation, but then arrest, and fail to grow. Analysis of gene expression patterns reveals that formation of the anterior visceral endoderm and the primitive streak is impaired in Cables2-deficient embryos. Tetraploid complementation analyses support the critical requirement of Cables2 in both the epiblast and visceral endoderm for progression of embryogenesis. In addition, we show that Cables2 physically interacts with a key mediator of the canonical Nodal pathway, Smad2, and augments its transcriptional activity. These findings provide novel insights into the essential role of Cables2 in the early embryonic development in mice.

81 Generation of presumptive domestic cat tetraploid embryos and its application for asynchronic complementation with diploid blastomeres

Tetraploid complementation has been extensively used to verify the pluripotency of stem cells and also for improving placenta formation when tetraploid embryos are aggregated synchronously or asynchronously with diploid (2n) embryos. Generation of tetraploid embryos can be achieved by the electric fusion of a 2-cell embryo. However, the optimal electric intensity pulse to generate tetraploid embryos has not been studied in the feline. The aims of this study were to (1) evaluate the optimal fusion conditions to achieve the highest fusion rate without affecting embryo developmental competence, (2) compare the in vitro development of synchronic and asynchronic aggregated domestic cat IVF embryos, and (3) assess pre-implantation development of embryos generated by asynchronic complementation of presumptive 1-cell tetraploid embryos with diploid blastomeres. Domestic cat cumulus-oocyte complexes were matured in vitro on 21% O2 in air at 38.5°C for 22h. The IVF embryos were generated by co-incubation of in vitro-matured oocytes with 2×106 motile spermatozoa mL−1 on 21% O2 in air at 38.5°C for 18 to 20h. After 24h of IVF, 2-cell embryos were selected. For Experiment 1, membrane fusion of 2-cell IVF embryos (n=164) was performed with two 30-ms DC pulses at different electric field (0.8, 2, 4, and 8 kV/cm) in fusion media (Mannitol, MgSO4, CaCl2, and polyvinyl alcohol). Presumptive fused embryos and nonfused were cultured in vitro in 50-µL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C (Pope et al. 2006 Methods in Molecular Biology 254, 227-244). Cleavage was determined 24h after pulse. For Experiment 2, zona pellucida-free IVF embryos (n=110) were synchronically (two 4-cell embryos) or asynchronically (one 4-cell embryo and one 2-cell embryo) aggregated in 1 microwell. For Experiment 3, 1-cell presumptive tetraploid embryo (2-cell fused embryo) was asynchronically complemented with a 4-cell embryo (n=38). For all experiments, blastocyst stage was evaluated at Day 8, and embryos presenting more than one structure per microwell were considered non-aggregated. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), and differences were considered significant at P<0.05. The highest fusion rates (30 and 46%) with the best developmental competence (31 and 46%) were observed with 4 and 8 Kv/cm electric pulses, respectively. Electric fusion did not affect the embryo developmental competence. We observed that synchronic and asynchronic complementation reached similar blastocysts rates (54 and 65%, respectively), indicating that both techniques are suitable for tetraploid embryo complementation. Finally, when presumptive tetraploid embryos were asynchronically complemented with diploid blastomeres, the high blastocyst rate (90%) was obtained from embryos that form only one structure (aggregated embryos). Further experiments will be performed to track the distribution of cells using mitotrackers after complementation using tetraploid IVF and diploid somatic cell nuclear transfer embryos.