Generation of germ cell-deficient pigs by NANOS3 knockout.

NANOS3 is an evolutionarily conserved gene expressed in primordial germ cells that is important for germ cell development. Germ cell deletion by NANOS3 knockout has been reported in several mammalian species, but its function in pigs is unclear. In the present study, we investigated the germline effects of NANOS3 knockout in pigs using CRISPR/Cas9. Embryo transfer of CRISPR/Cas9-modified embryos produced ten offspring, of which one showed wild-type NANOS3 alleles, eight had two mutant NANOS3 alleles, and the other exhibited mosaicism (four mutant alleles). Histological analysis revealed no germ cells in the testes or ovaries of any of the nine mutant pigs. These results demonstrated that NANOS3 is crucial for porcine germ cell production.

Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation.

Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 µm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 µm) as compared to those from the control group (178.9 ± 9.0 µm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles.

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.

alpha1,3-Galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences and cre-expressing adenovirus.

BACKGROUND: Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine alpha1,3-galactosyltransferase (alpha1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and alpha1,3GT-gene knockout cattle. METHODS: alpha1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using alpha1,3GT fetal fibroblasts, and one alpha1,3GT knockout calf was generated but died shortly after birth (day 287). RESULTS: Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range. CONCLUSION: The alpha1,3GT knockout- and antibiotic-resistance gene free (alpha1,3GT(-/-)neo-) cells could be cloned normally. Thus, cloned cattle from alpha1,3GT(-/-) neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.

Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle.

Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3(-/-)) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3(+/+)) were injected into NANOS3(-/-) Wagyu embryos. Subsequently, exogenous germ cells (NANOS3(+/+)) were identified in the NANOS3(-/-) ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies.

Production of hepatitis E virus-like particles presenting multiple foreign epitopes by co-infection of recombinant baculoviruses.

Hepatitis E virus (HEV) causes not only endemics via a fecal-oral route but also sporadic cases via zoonotic transmission or blood transfusion. HEV-like particles (HEV-LP) produced by using a baculovirus expression system are considered a candidate for mucosal vaccines for HEV infection. In this study, we attempted to produce a chimeric HEV-LP presenting various foreign epitopes on its surface. Expression of the recombinant capsid proteins carrying a myc- or FLAG-tag inserted between amino acid residues 488 and 489, which are located in the exterior loop on the protruding domain of the HEV capsid, resulted in the production of recombinant HEV-LP. Although expression of the recombinant capsid protein carrying the HA-tag inserted at the same site failed to produce any particles, co-expression with the myc-tagged capsid protein successfully yielded a chimeric HEV-LP consisting of both recombinant capsid proteins. Immunoprecipitation analyses confirmed that the chimeric particles present these foreign epitopes on the surface. Similar results were obtained for the expression of the recombinant capsid proteins carrying neutralizing epitopes of Japanese encephalitis virus. These results suggest the chimeric HEV-LP system provides a novel vaccine carrier that can accommodate multiple neutralizing epitopes on its surface.

Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization.

The mammalian fertilization process takes place in a complex microenvironment within the female genital tract. A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), has been identified in oviductal fluid from various mammalian species, including humans. Although OGP is widely believed to be involved in the process of mammalian fertilization, including spermatozoon function and gamete interactions, based on experimental results obtained in vitro, its physiological significance remains controversial. The present study established OGP gene-null ( ogp (-/-)) mice, and primarily characterized their reproductive properties to study the physiological function(s) of OGP. Results obtained from studies using an in vivo or in vitro system showed that the fertility of ogp (-/-) females was within normal limits. These results indicate that OGP is not essential for the process of in vivo fertilization, at least in mice.

Heterozygous disruption of the alpha1,3-galactosyltransferase gene in cattle.

BACKGROUND: Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the alpha1,3-galactosyltransferase (alpha1,3-GT) gene in cattle. METHODS: The alpha1,3-GT gene of the Japanese Black cow (JBC) was used to construct pGT-6, a targeting vector for the bovine alpha1,3-GT gene, and pGT-6 was introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Four polymerase chain reaction (PCR)-positive colonies were obtained from 797 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the alpha1,3-GT locus in one of the four colonies. Nuclear transfer was performed, and the four embryos were transferred to a heifer. RESULTS: To establish fetal fibroblasts that were heterozygously disrupted at the alpha1,3-GT locus, one of the fetuses was recovered at 5 weeks of pregnancy, and PCR and Southern blot analysis of the fetal fibroblasts established from it showed definite homologous recombination of the alpha1,3-GT gene. CONCLUSIONS: Heterozygous knockout of the alpha1,3-GT gene was performed in JBC, and production of a homozygous alpha1,3-GT knockout JBC by a second round of targeting 906htGT is currently in progress. The technique described here can be applied to disruption of other genes in cattle.

Activation of Ca2+ Transport System of Sea Urchin Sperm by High External pH: 220 kD Membrane Glycoprotein is Involved in the Regulation of the Ca2+ Entry: (sea urchin sperm/external pH/intracellular pH/Ca2+ entry/acrosome reaction).

When sperm of the sea urchin, Hemicentrotus pulcherrimus, were exposed to high pH (9.0) sea water, they showed large increases in intracellular Ca2+ ([Ca2+ ]i) and pH (pHi) and underwent the acrosome reaction (AR) without the aid of the egg jelly. Not only [Ca2+ ]i increase but also pHi rise did not occur under Ca2+ -free conditions. Both the increases in [Ca2+ ]i and pHi and the AR by high pH were inhibited by a Ca2+ channel blockers, verapamil and nisoldipine, and by a lectin, wheat germ agglutinin (WGA) which interacts with a 220 kD membrane glycoprotein of sperm. These reagents inhibited also the AR by the egg jelly. The inhibitory effects of WGA were immediately canceled by the addition of N-acetyl-D-glucosamine, a sugar which is known to remove WGA from its binding site. These results suggest that 1) the same Ca2+ transport system is activated by high external pH and the egg jelly, 2) increase in [Ca2+ ]i is prerequisite for the stimulation of the H+ -efflux system(s) and 3) the 220 kD WGA-binding membrane protein functions as a regulator protein of Ca2+ transport system.

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. II. Antibody against WGA-Binding Protein Induces the Acrosome Reaction: (WGA-binding protein/sea urchin/sperm/acrosome reaction).

We obtained a polyclonal antibody against the WGA-binding protein (WGAbp) of Strongylocentrotus intermedius sperm, which is a membrane glycoprotein of 260 kD under non-reducing condition. Anti-WGAbp antibody induced increases in both intracellular Ca2+ ([Ca2+ ]i) and intracellular pH (pHi), resulting in the onset of the AR. The increases in [Ca2+ ]i and pHi required extracellular Ca2+ and Na+ , respectively, and were suppressed by the pretreatment with WGA, resulting in the inhibition of the AR. Anti-WGAbp antibody-induced AR was inhibited also by lowered extracellular pH. elevated K+ , removal of Na+ from seawater and the treatment with verapamil, a Ca2+ channel inhibitor. These inhibitory conditions are identical with those of the egg jelly-induced AR. Monovalent Fab fragments from anti-WGAbp antibody also induced the AR at relatively high concentration. These results suggest that the WGAbp on the sperm plasma membrane is involved in the regulation of Ca2+ influx and Na+ /H+ exchange associated with the AR of S. intermedius sperm. It is a strong candidate for the receptor of the AR-inducing substance in the egg jelly.

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. I. WGA Affects the Ion Fluxes Associated with the Acrosome Reaction: (sea urchin/acrosome reaction/wheat germ agglutinin/ion flux/species specificity).

The lectin wheat germ agglutinin (WGA) inhibited the egg jelly-induced acrosome reaction (AR) of sperm of the sea urchin, Strongylocentrotus intermedius. Fluorescein-conjugated WGA applied to sperm bound to the acrosomal region, to the midpiece, and to the tip of the flagellum. These effects were not observed in the presence of N-acetly-D-glucosamine. When the egg jelly was replaced by artificial AR inducers such as A23187 or nigericin, the AR was not inhibited by WGA. Results obtained using a Ca2+ indicator fura-2, a pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) and a membrane potential sensitive dye 3,3'-dipentyl 2,2'-dioxacarbocyanine [diO-C5 (3)] showed that WGA suppresses the egg jelly-induced influx of Ca2+ and slightly suppresses the efflux of H+ caused by the egg jelly, whereas the depolarization of the plasma membrane by the egg jelly is remarkably amplified by the treatment with WGA. These results suggest that WGA affects the regulatory system of the ion fluxes associated with the AR. The target protein of WGA (WGA-binding protein) was a membrane glycoprotein of 260 kD under non-reducing condition.

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa: (FSG/SAP-I/[Ca2+ ]i /pHi ).

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pHi ) and Ca2+ ([Ca2+ ]i ) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus. FSG and/or SAP-I induced elevations of [Ca2+ ]; and pHi in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca2+ ]i or pHi of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca2+ ]i and pHi , At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca2+ ]i , although SAP-I elevated the pHi , its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K+ -channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca2+ ]i and pHi by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca2+ and H+ transport systems.

Generation of recombination activating gene-1-deficient neonatal piglets: a model of T and B cell deficient severe combined immune deficiency.

Although severe combined immune deficiency (SCID) is a very important research model for mice and SCID mice are widely used, there are only few reports describing the SCID pig models. Therefore, additional research in this area is needed. In this study, we describe the generation of Recombination activating gene-1 (Rag-1)-deficient neonatal piglets in Duroc breed using somatic cell nuclear transfer (SCNT) with gene targeting and analysis using fluorescence-activated cell sorting (FACS) and histology. We constructed porcine Rag-1 gene targeting vectors for the Exon 2 region and obtained heterozygous/homozygous Rag-1 knockout cell colonies using SCNT. We generated two Rag-1-deficient neonatal piglets and compared them with wild-type neonatal piglets. FACS analysis showed that Rag-1 disruption causes a lack of Immunoglobulin M-positive B cells and CD3-positive T cells in peripheral blood mononuclear cells. Consistent with FACS analysis, histological analysis revealed structural defects and an absence of mature lymphocytes in the spleen, mesenteric lymph node (MLNs), and thymus in Rag-1-deficient piglets. These results confirm that Rag-1 is necessary for the generation of lymphocytes in pigs, and Rag-1-deficient piglets exhibit a T and B cell deficient SCID (T-B-SCID) phenotype similar to that of rodents and humans. The T-B-SCID pigs with Rag-1 deficiency generated in this study could be a suitably versatile model for laboratory, translational, and biomedical research, including the development of a humanized model and assessment of pluripotent stem cells.

Growth, antrum formation, and estradiol production of bovine preantral follicles cultured in a serum-free medium.

The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.