Publisher Correction: Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle.

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Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle.

Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

B-box and SPRY domain containing protein (BSPRY) is associated with the maintenance of mouse embryonic stem cell pluripotency and early embryonic development.

Mouse embryonic stem (ES) cells consist of heterogeneous populations with differing abilities to proliferate and differentiate. We previously demonstrated that the expression level of platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31 was positively correlated with the undifferentiated state of mouse ES cells. In order to screen for a novel gene(s) involved in ES cell pluripotency, we performed an oligo microarray analysis and identified that B-box and SPRY domain containing protein (BSPRY) was expressed at high levels in PECAM1-positive cells. Two splice isoforms of BSPRY, BSPRY-1 and BSPRY-2, were expressed in undifferentiated ES cells and in blastocysts. Knockdown of BSPRY-1/2 in ES cells significantly reduced the number of undifferentiated colonies and caused increased expression of primitive ectoderm marker gene Fgf5. The overexpression of BSPRY-2 reciprocally increased the number of undifferentiated ES cells in the presence of LIF. Similarly, injection of BSPRY-1/2 siRNAs into 2-cell embryos caused developmental retardation and degeneration of embryos, and a significant decrease in the number of cells, especially in the inner cell mass (ICM), was observed at the blastocyst stage. Furthermore, microinjection of a BSPRY-1 expression vector into pronuclear stage embryos resulted in an increase in the hatching blastocysts rate after 120 h of culture. These results suggest that BSPRY-1 and BSPRY-2 are associated with both ES cell pluripotency and early embryonic development.

Expression and characterization of novel ovine orthologs of bovine placental prolactin-related proteins.

BACKGROUND: The prolactin-related proteins (PRPs) are non-classical placental-specific members of the prolactin/growth hormone family. Among ruminants, they are expressed in the cotyledonary villi of cattle and goat. We investigated placental PRP in sheep in order to gain a comprehensive understanding of the function and evolution of these molecules. We also examined the sequence properties, expression and lactogenic activation of the cloned genes. RESULTS: We cloned two novel ovine PRPs, named oPRP1 and oPRP2. oPRP2 had a typical PRP sequence similar to bovine PRP1 (bPRP1). oPRP1 had a short sequence identical with bovine or caprine type PRP but the reading frame was shifted. Both oPRPs were expressed in trophoblast giant binucleate cells (BNC) as in cattle and goat. oPRP1 expression declined from the early to the middle stage of gestation. In contrast, oPRP2 expression remained constant throughout the gestation period. oPRP2 was translated to form a mature protein in a mammalian cell expression system. Western blotting showed a molecular mass of 35 kDa for the FLAG-tag fusion oPRP2 protein. This recombinant protein and bPRP1 were bioassayed using Nb2 lymphoma cells; it was confirmed that neither ruminant PRP had lactogenic activity because the Nb2 lymphoma cells did not proliferate. CONCLUSION: We have identified two novel PRPs, oPRP1 and oPRP2, in ovine placenta. Both these ovine PRPs were localized and quantitatively expressed in BNC. Absence of lactogenic activity was confirmed for the oPRP2 molecule. It is anticipated that novel and known ruminant PRPs have common functions, except for lactogenic activity.

Gene expression profiling of mouse embryonic stem cell subpopulations.

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.

Differences in gene expression patterns between somatic cell nuclear transfer embryos constructed with either rabbit granulosa cells or their derivatives.

Successful production of offspring by somatic cell nuclear transfer (SCNT) is affected by the nature of the donor cells used. The purpose of this study was to determine whether characteristic changes induced in donor cells by culture conditions influenced gene expression patterns in the resultant SCNT embryos. Rabbit granulosa cells (rGC) were cultured under different conditions, either with or without hCG, and the two derivative cell types obtained (named respectively cGC+ and cGC-) were used as donor cells for SCNT. There were characteristic differences between fresh rGC and the two derivative cell types: p450scc expression and progesterone secretion were both higher in cGC+ than in cGC-; expression of bmp4 and fgfr2 was decreased in cGC+ and cGC- compared with rGC; and cGC+ and cGC- cell types gained collagenIV expression. Use of fresh rGC, or cGC+ and cGC- derivative cells, did not alter either the developmental potencies of SCNT oocytes or cell numbers at the blastocyst stage. The expression patterns of four genes (bmp4, fgfr2, gata4, oct3/4) in SCNT embryos and in fertilized embryos were analyzed by quantitative RT-PCR. We found that oct3/4 was expressed in all embryos. The expression patterns of the other three genes showed considerable variation between the different types of embryo: bmp4 was found in most fertilized embryos but only some of rGC and none of cGC+ and cGC- derived SCNT embryos; fgfr2 was present in fertilized embryos but was present in some rGC and cGC- NT embryos and in all cGC+ NT embryos; gata4 was not expressed in fertilized embryos but was present in a few rGC and cGC+ NT embryos and in most cGC- NT embryos. Our results suggest that the gene expression patterns in SCNT embryos derived from granulosa donor cells are affected by characteristic changes to the cells during in vitro culture.

Embryonic stem cells expressing both platelet endothelial cell adhesion molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in a chimeric embryo.

We examined the expression of cell-surface markers on subpopulations of mouse embryonic stem (ES) cells to identify those that were associated with cells that had the highest pluripotency. Flow cytometry analysis revealed a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic antigen (SSEA)-1 in ES cells. Almost all SSEA-1+ cells expressed a high level of PECAM- 1, and reversible repopulation was observed between PECAM- 1+SSEA-1+ and PECAM-1+SSEA-1- cells. The ES cells carrying the lacZ gene were sorted into three subpopulations: PECAM- 1-SSEA-1-, PECAM-1+SSEA-1-, and PECAM-1+SSEA-1+. Quantitative reverse transcription-polymerase chain reaction revealed a low level of Oct3/4 mRNA expression and an elevation in differentiation maker gene expression in PECAM-1- cells. To compare the pluripotency of these three subpopulations, a single cell from each was injected into eight-cell embryo and ES cells identified at later stages by X-gal staining. At the blastocyst stage, PECAM-1+ SSEA-1+/- cells were found to have differentiated into epiblast cells in high numbers. In contrast, PECAM- 1- cell derivatives localized in the primitive endoderm or trophectoderm. At 6.0-7.0 days post coitum, many PECAM-1+SSEA- 1+ cells were found in the epiblast, but few beta-gal+ cells were detected in any regions of embryos that were injected with cells from the other two populations. These results showed that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos.

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo.

To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer.

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.