In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System.

The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.

Germline deletion of Cdyl causes teratozoospermia and progressive infertility in male mice.

Chromodomain Y (CDY) is one of the candidate genes for male dyszoospermia related to Y chromosome microdeletion (YCM). However, the function of CDY in regulating spermatogenesis has not been completely determined. The mouse Cdyl (CDY-like) gene is the homolog of human CDY. In the present study, we generated a germline conditional knockout (cKO) model of mouse Cdyl. Significantly, the CdylcKO male mice suffered from the defects in spermatogonia maintenance and spermatozoon morphogenesis, demonstrating teratozoospermia and a progressive infertility phenotype in early adulthood. Importantly, patterns of specific histone methylation and acetylation were extensively changed, which disturbed the transcriptome in CdylcKO testis. Our findings indicated that Cdyl is crucial for spermatogenesis and male fertility, which provides novel insights into the function of CDY gene, as well as the pathogenesis of YCM-related reproductive failure.

Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System.

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.

Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.

STUDY QUESTION: Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? SUMMARY ANSWER: Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. WHAT IS KNOWN ALREADY: The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. STUDY DESIGN, SIZE, DURATION: Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. MAIN RESULTS AND THE ROLE OF CHANCE: The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. WIDER IMPLICATIONS OF THE FINDINGS: Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. STUDY FUNDING/COMPETING INTERESTS: The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.

Paternal Psychological Stress Reprograms Hepatic Gluconeogenesis in Offspring.

Both epidemiologic and experimental animal studies demonstrate that chronic psychological stress exerts adverse effects on the initiation and/or progression of many diseases. However, intergenerational effects of this environmental information remains poorly understood. Here, using a C57BL/6 mouse model of restraint stress, we show that offspring of stressed fathers exhibit hyperglycemia due to enhanced hepatic gluconeogenesis and elevated expression of PEPCK. Mechanistically, we identify an epigenetic alteration at the promoter region of the Sfmbt2 gene, a maternally imprinted polycomb gene, leading to a downregulation of intronic microRNA-466b-3p, which post-transcriptionally inhibits PEPCK expression. Importantly, hyperglycemia in F1 mice is reversed by RU486 treatment in fathers, and dexamethasone administration in F0 mice phenocopies the roles of restraint stress. Thus, we provide evidence showing the effects of paternal psychological stress on the regulation of glucose metabolism in offspring, which may have profound implications for our understanding of health and disease risk inherited from fathers.

Ggnbp2 Is Essential for Pregnancy Success via Regulation of Mouse Trophoblast Stem Cell Proliferation and Differentiation.

The Ggnbp2 null mutant embryos died in utero between Embryonic Days 13.5 to 15.5 with dysmorphic placentae, characterized by excessive nonvascular cell nests consisting of proliferative trophoblastic tissue and abundant trophoblast stem cells (TSCs) in the labyrinth. Lethality of Ggnbp2 null embryos was caused by insufficient placental perfusion as a result of remarkable decreases in both fetal and maternal blood vessels in the labyrinth. These defects were accompanied by a significant elevation of c-Met expression and phosphorylation and its downstream effector Stat3 activation. Knockdown of Ggnbp2 in wild-type TSCs in vitro provoked the proliferation but delayed the differentiation with an upregulation of c-Met expression and an enhanced phosphorylation of c-Met and Stat3. In contrast, overexpression of Ggnbp2 in wild-type TSCs exhibited completely opposite effects compared to knockdown TSCs. These results suggest that loss of GGNBP2 in the placenta aberrantly overactivates c-Met-Stat3 signaling, alters TSC proliferation and differentiation, and ultimately compromises the structure of placental vascular labyrinth. Our studies for the first time demonstrate that GGNBP2 is an essential factor for pregnancy success acting through the maintenance of a balance of TSC proliferation and differentiation during placental development.

Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1, 2-propanediol.

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes.

The Oct4 promoter-EGFP transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells.

The rabbit has long been used as a laboratory animal model for developing reproductive and stem cell-related technologies, as well as for studying human disease. The Oct4 transcription factor plays a crucial role in the maintenance and regulation of pluripotency in embryos and stem cells. We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein (EGFP) under the control of the rabbit Oct4 promoter (prOG) and transfected it into E14 mouse stem cells and rabbit ESCs. In addition, prOG transgenic fibroblasts were derived and prOG transgenic rabbits were produced by somatic cell nuclear transfer (SCNT). The pattern of expression of ectopic EGFP was similar in E14 mouse stem cells whether under the control of the rabbit (prOG) or mouse Oct4 promoter (pmOG). EGFP expression was observed in rabbit ESCs following prOG transfection. Both prOG transgenic SCNT embryos and F1 prOG transgenic embryos derived from adult transgenic rabbits expressed green fluorescence at the morula and blastocyst stages. EGFP was clearly detected in gonads isolated from fetuses at 27 dpc. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, and for investigating rabbit embryo development.

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts.

Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts were transfected with a pEGFP-C1 plasmid using lipofectamine() 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than 9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits.

Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries: a new approach to autologous stem cell therapy.

OBJECTIVE: To evaluate whether cryopreserved newborn mouse ovaries can generate sufficient numbers of parthenogenetic mouse embryonic stem (pmES) cells for autologous stem cell therapy. DESIGN: Prospective study. SETTING: Reproductive clinic of Xinhua Hospital in Shanghai. ANIMAL(S): Kunming, C57BL/6J, BALB/c, and NOD-SCID mice. INTERVENTION(S): Cryopreserved newborn mouse ovaries were thawed, grafted into immunodeficient mice, treated with pregnant mare serum gonadotropin to promote follicular maturation, and collected oocytes activated in vitro to generate parthenogenetic embryonic stem cells. MAIN OUTCOME MEASURE(S): Preimplantation development and stem cell characterization. RESULT(S): This new protocol yielded a large number of oocytes from cryopreserved ovaries over a long period. These oocytes were used to derive pmES cell lines, which expressed embryonic stem cell-specific markers and differentiated into embryoid bodies in vitro and teratomas in vivo. The pmES cell line was propagated in an undifferentiated state for more than 30 passages and maintained a diploid karyotype. CONCLUSION(S): The pmES cells lines established by our protocol exhibited the same degree of pluripotency as standard embryonic stem cell lines. This approach may be used for exploring autologous stem cell therapies.

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1x10(5) ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1x10(6) ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm2) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm2). These findings provided fresh insight into the neural induction of mouse ES cells.

Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis.

Somatic cell nuclear transfer (SCNT) and parthenogenesis are alternative forms of reproduction and development, building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin, respectively. In the preceding paper (Sun F, et al., Cell Res 2007; 17:117-134.), we showed that an "erase-and-rebuild" strategy is used in normal development to transform the maternal gene expression profile to a zygotic one. Here, we investigate if the same strategy also applies to SCNT and parthenogenesis. The relationship between chromatin and chromatin factors (CFs) during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein assays. Results from these studies indicated that soon after nuclear transfer, a majority of CFs dissociated from somatic nuclei and were redistributed to the cytoplasm of the egg. The erasure process in oogenesis is recaptured during the initial phase in SCNT. Most CFs entered pseudo-pronuclei shortly after their formation. In parthenogenesis, all parthenogenotes underwent normal oogenesis, and thus had removed most CFs from chromosomes before the initiation of development. The CFs were subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized embryos. Based on these data, we conclude that the "erase-and-rebuild" process observed in normal development also occurs in SCNT and in parthenogenesis, albeit in altered fashions. The process is responsible for transcription reprogramming in these procedures. The "erase" process in SCNT is compressed and the efficiency is compromised, which likely contribute to the developmental defects often observed in nuclear transfer (nt) embryos. Furthermore, results from this study indicated that the cytoplasm of an egg contains most, if not all, essential components for assembling the zygotic program and can assemble them onto appropriate diploid chromatin of distinct origins.