Oocyte activation with phospholipase Cζ mRNA induces repetitive intracellular Ca2+ rises and improves the quality of pig embryos after intracytoplasmic sperm injection.

For the intracytoplasmic sperm injection (ICSI) procedure in pigs, an electrical pulse (EP) has been used as an effective method for oocyte stimulation, but unlike sperm, EP is unable to induce Ca2+ oscillations. In this study, we investigated the effects of generating artificial Ca2+ oscillations with phospholipase Cζ (PLCζ) mRNA, a candidate sperm factor, on fertilization, embryonic development, and gene expression after ICSI. Firstly, the concentration of PLCζ mRNA of a fixed volume (1.0 pl) that would induce a pattern of Ca2+ rise similar to that of in vitro fertilized (IVF) sperm was examined and determined to be 300 ng/µl. Secondly, the effects of oocyte stimulation methods on fertilization and embryonic development were investigated. ICSI-oocytes were activated by EP (EP group) or by PLCζ mRNA (PLCζ group). Furthermore, IVF-oocytes (IVF group) and ICSI-oocytes with and without an injection of buffer (buffer and untreated groups, respectively) were used as controls. It was found that the rates of normal fertilization in the PLCζ and EP groups were significantly higher than those in the buffer and untreated groups. The blastocyst formation rates did not differ among the groups. The embryo quality in the EP group was inferior to those in the PLCζ and IVF groups. Additionally, the expression level of a proapoptosis-related gene (Caspase-3) in the EP group was significantly higher than those in the PLCζ and IVF groups. Our data suggest that oocyte activation by PLCζ mRNA has the effect of improving embryo quality.

Effect of Growth Hormone Receptor Deficiency on Androgen-Associated Gene Expression of Hepatic Drug Metabolizing Enzymes and Drug Transporters in Pigs.

Growth hormone receptor (GHR)-deficient pigs were generated using the CRISPR/Cas9 system to investigate the involvement of GHR-mediated growth hormone (GH) signaling in androgen-associated gene expression of hepatic drug metabolizing enzymes (DMEs) and drug transporters. We initially confirmed that no wild-type GHR mRNA was present in GHR-/- (GHR-KO) pigs; in addition, as previously reported, those pigs exhibited decreases in body weight and serum insulin-like growth factor-1 concentration and an increase in serum GH concentration compared with the levels in GHR-/+ and GHR+/+ pigs with a wild-type GHR mRNA. The real-time RT-PCR results on the mRNA levels of hepatic DMEs and drug transporters in the GHR-KO pigs and the pigs with a wild-type GHR mRNA revealed that, among the examined hepatic DMEs, the mRNA levels of CYP1A2, CYP2A19, sulfotransferase (SULT) 1A1, and SULT2A1 were higher in GHR-KO pigs than in the pigs with a wild-type GHR mRNA, whereas the opposite trend was observed for the mRNA level of uridine 5'-diphospho-glucuronosyltransferase 1A6. No such significant differences in the mRNA levels of three hepatic drug transporters including multidrug resistance protein 1 were observed. In addition, the mRNA level of hepatic cut-like homeobox 2 (CUX2), which is expressed in an androgen-dependent manner and associated with the hepatic mRNA expression of several DMEs, was significantly decreased in GHR-KO pigs. The present findings strongly suggest that not only serum androgen but also GHR-mediated GH signaling contributes to the mRNA expression of several DMEs and CUX2, but not transporters, in the pig liver.

Establishment of porcine nuclear transfer-derived embryonic stem cells using induced pluripotent stem cells as donor nuclei.

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success.

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs.

In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca2+ signaling, also known as Ca2+ oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes. Therefore, we compared the patterns of Ca2+ oscillation, the efficiency of oocyte activation and normal fertilization, and embryo development to the blastocyst stage among in vivo- or in vitro-matured oocytes after ICSI or in vitro fertilization (IVF). Unexpectedly, we found that the pattern of Ca2+ oscillation, such as the frequency and amplitude of Ca2+ rises, in oocytes after ICSI was similar to that in oocytes after IVF, irrespective of the oocyte source. However, half of the oocytes failed to become activated after ICSI and showed no Ca2+ oscillation. Moreover, the embryonic development of normal fertilized oocytes was reduced when in vitro-matured oocytes were used, irrespective of the fertilization method employed. These findings suggest that low embryo production efficiency after ICSI is attributable mainly to poor developmental ability of in vitro-matured oocytes and a lack of Ca2+ oscillation, rather than the pattern of oscillation.

Characterization of actions of octanoate on porcine preadipocytes and adipocytes differentiated in vitro.

Octanoate is used to induce adipogenic differentiation and/or lipid accumulation in preadipocytes of domestic animals. However, information on detailed actions of octanoate and the characteristics of octanoate-induced adipocytes is limited. The aim of this study was to examine these issues by comparing the outcomes of the effects of octanoate with those of rosiglitazone, which is a well-defined activator of peroxisome proliferator-activated receptor (PPAR)-γ. The adipocytes that were differentiated with 5mM of octanoate had dispersed and diversely sized lipid droplets compared to those that were differentiated with 1 µM of rosiglitazone. The gene expression levels of adiponectin, glycerol-3-phosphate dehydrogenase, perilipin 1, and perilipin 4 were much higher in the adipocytes that were differentiated with rosiglitazone than in those differentiated with octanoate, while the gene expression levels of lipoprotein lipase and perilipin 2 were decreased in rosiglitazone-differentiated adipocytes compared to octanoate-differentiated adipocytes. However, the expressions of aP2 and CD36 genes were comparably induced. Luciferase reporter assays revealed that PPAR and liver-X-receptor activities were upregulated by octanoate more effectively than by rosiglitazone. Overall, these results suggested that the action of octanoate was complicated and may be dependent on the targeted genes and cellular status.

In vitro fertilization and development of porcine oocytes matured in follicular fluid.

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10(6) cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.

Production of cloned pigs expressing human thrombomodulin in endothelial cells.

For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.

Ploidy assessment of porcine haploid and diploid parthenogenetic embryos by fluorescent in situ hybridization detecting a chromosome 1-specific sequence, Sus scrofa Mc1 satellite DNA.

The aim of the present study was to examine the feasibility of fluorescent in situ hybridization (FISH) for detecting a chromosome 1-specific sequence as a means of assessing the ploidy of porcine parthenotes. In vitro-matured oocytes with the first polar body (PB) were electrically activated; some were treated with cytochalasin B to prevent second PB extrusion (1PB embryos), and the others extruded the second PB (2PB embryos). At the 2-cell stage, one and two FISH signals were detected in each nucleus of 2PB and 1PB embryos, respectively. Almost all cells of blastocysts derived from 1PB embryos retained two signals. In contrast, cells of blastocysts derived from 2PB embryos had two signals. These data demonstrate that FISH analysis allows precise ploidy assessment of porcine parthenogenetic embryos, hence providing a practical means of detecting ploidy transition during parthenogenetic embryogenesis.

Pathological findings after third- and second-generation everolimus-eluting stent implantations in coronary arteries from autopsy cases and an atherosclerotic porcine model.

Pathological changes after third-generation drug-eluting stent implantation remain unclear. We compared the tissue responses of coronary arteries after the implantation of third-generation abluminal biodegradable-polymer everolimus-eluting stent (3rd EES) and second-generation durable-polymer EES (2nd EES) using autopsy specimens and an atherosclerotic porcine model. We compared the histology of stented coronary arteries obtained by autopsy performed 1-10 months after 3rd EES (n (number of cases) = 4, stent-implanted period of 3-7 months) and 2nd EES (n (number of cases) = 9, stent-implanted period of 1-10 months) implantations. The ratio of covered stent struts was higher with 3rd EESs than with 2nd EESs (3rd; 0.824 ± 0.032 vs. 2nd; 0.736 ± 0.022, p = 0.035). Low-density lipoprotein receptor knockout minipigs were stented with 3rd or 2nd EES in the coronary arteries and the stented regions were investigated. The fibrin deposition around the 2nd EES was more prominent. Additionally, higher density of smooth muscle cells was confirmed after the 3rd EES implantation. Pathological examination after the 3rd EES demonstrated a combination of less fibrin deposition and more rapid acquisition of well-developed neointima as compared to the 2nd EES at autopsy and the atherosclerotic porcine model.

Intraoperative laparoscopic detection of sentinel lymph nodes with indocyanine green and superparamagnetic iron oxide in a swine gallbladder cancer model.

Mapping of sentinel lymph nodes (SLNs) can enable less invasive surgery. However, mapping is challenging for cancers of difficult-to-access visceral organs, such as the gallbladder, because the standard method using radioisotopes (RIs) requires preoperative tracer injection. Indocyanine green (ICG) and superparamagnetic iron oxide (SPIO) have also been used as alternative tracers. In this study, we modified a previously reported magnetic probe for laparoscopic use and evaluated the feasibility of detecting SLNs of the gallbladder using a laparoscopic dual tracer method by injecting ICG and SPIO into five swine and one cancer-bearing swine. The laparoscopic probe identified SPIO nanoparticles in the nodes of 4/5 swine in situ, the magnetic field counts were 2.5-15.9 µT, and fluorescence was detected in SLNs in all five swine. ICG showed a visual lymph flow map, and SPIO more accurately identified each SLN with a measurable magnetic field quite similar to the RI. We then developed an advanced gallbladder cancer model with lymph node metastasis using recombination activating gene 2-knockout swine. We identified an SLN in the laparoscopic investigation, and the magnetic field count was 3.5 µT. The SLN was histologically determined to be one of the two metastatic lymph nodes. In conclusion, detecting the SLNs of gallbladder cancer in situ using a dual tracer laparoscopic technique with ICG and SPIO was feasible in a swine model.

Organogenesis of Ileal Peyer's Patches Is Initiated Prenatally and Accelerated Postnatally With Comprehensive Proliferation of B Cells in Pigs.

Morphogenesis and differentiation of organs is required for subsequent functional maturation. The morphological features of Peyer's patches vary among species. In pigs, they develop extensively in the ileum as ileal Peyer's patches (IPPs). However, the role of IPPs in the porcine immune system remains to be elucidated because of a lack of complete understanding of IPP organogenesis. Results of the present study revealed that development of porcine IPPs is initiated prenatally between embryonic days 76 and 91. The process of IPP organogenesis is concomitant with increased transcriptional patterns of CXCL13 and CCL19. IPPs undergo further development postnatally by forming central, marginal, and subepithelial zones. Importantly, a large number of proliferating B cells and apoptotic cells are found in porcine IPPs postnatally, but not prenatally. The expression level of IgM in proliferating B cells depends on the zone in which distinct B cells are separately localized after birth. Specifically, IgM+ cells are predominantly found in the central zone, whereas IgM-/low cells are abundant in the marginal zone. Importantly, the cellular feature of IPPs differs from that of mesenteric lymph nodes (MLNs) where such distinct zones are not formed both prenatally and postnatally. Our findings suggest that IPPs (not MLNs) in postnatal pigs are involved in complementing functions of the primary lymphoid tissue that promotes the differentiation and maturation of B cells.

Effect of pemafibrate (K-877), a novel selective peroxisome proliferator-activated receptor α modular (SPPARMα), in atherosclerosis model using low density lipoprotein receptor knock-out swine with balloon injury.

BACKGROUND: Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that has key roles of lipid metabolism and inflammation. The PPARα may affects the initiation and progression of atherosclerosis by reducing inflammatory responses. Pemafibrate (K-877) is a novel selective PPARα modulator (SPPARMα), which was designed to possess higher PPARα potency and selectivity than existing PPARα agonists. The aim of this study is to evaluate the effect of pemafibrate on vascular response in coronary atherosclerosis model using low density lipoprotein receptor knock-out (LDLR-KO) pigs with balloon injury. METHODS AND RESULTS: Ten LDLR-KO pigs were randomly allocated to two groups [pemafibrate (n = 5) and control (n = 5)] and fed with a diet containing 2.0% cholesterol and 20% lard throughout the study. Balloon injury was created in 40 coronary segments two weeks after starting the oral administration of pemafibrate or placebo. Necropsy was conducted 8 weeks later. Coronary artery sections were reviewed to evaluate lesion progression and the mRNA expression levels for C-Jun, NFκ B, CCL2, CCR7, CD163 and MMP9 determined using real-time RT-PCR. LDL cholesterol at baseline was about 700 mg/dL. The mean ratio of macrophages to plaque area was significantly lower in pemafibrate group compared with control one (7.63±1.16 vs 14.04±4.51, P = 0.02) whereas no differences were observed in intimal area between groups. The mRNA levels of C-Jun, NFκB and MMP9 were significantly decreased in pemafibrate group. CONCLUSIONS: Pemafibrate was associated with inhibition of inflammatory responses in coronary artery atherosclerosis model using LDLR-KO swine with balloon injury.

Elucidation of the Effects of a Current X-SCID Therapy on Intestinal Lymphoid Organogenesis Using an In Vivo Animal Model.

BACKGROUND & AIMS: Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID. METHODS: We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID. RESULTS: We demonstrated that X-SCID pigs completely lacked Peyer's patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG. CONCLUSIONS: These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo.

Successful cross-breeding of cloned pigs expressing endo-beta-galactosidase C and human decay accelerating factor.

BACKGROUND: For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of alphaGal expression by its digestion enzyme, endo-beta-galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross-breeding of pigs expressing EndoGalC and hDAF. METHODS: After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace x Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross-breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction. RESULTS: Well-designed cloned pigs were produced by cross-breeding. alphaGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild-type pigs. hDAF expression reached about 10- to 70-fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti-pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week. CONCLUSIONS: Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of alphaGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.

Fetal and early postnatal development of the porcine tonsils of the soft palate.

Tonsils are mucosa-associated lymphoid tissues located at the openings of the gastrointestinal and respiratory tracts, which play a key role in the surveillance of inhaled or ingested pathogens and can concurrently be reservoirs of infectious agents. Therefore, tonsils are important for the immunology and hygiene management of domestic animals, including pigs. However, the process of their fetal developmental has been poorly described, at least in part, because rodents lack tonsils. Therefore, we performed a histological analysis of porcine tonsils of the soft palate from 60 to 100 days of gestation (DG) and from 2 to 14 days post partum (DP). This analysis showed that lymphoid aggregations first appear at DG65, gradually develop during the fetal stage, and expand after birth. In addition, the mRNA expression of chemokine genes involved in lymphoid aggregation and localization was analyzed. CCL19 expression showed the most marked increase and a sharp peak after birth. CCL21 expression changed moderately but showed an interesting bimodal pattern. CXCL13 expression steadily increased throughout the study period. Thus, we demonstrated the mRNA expression of chemokine characteristically changed accompanying tonsillar development.

Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation.

Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.

Establishment of a model of sentinel lymph node metastasis using immunodeficient swine.

Lymph node metastasis occurs via the migration of cancer cells through the lymphatic system. Sentinel lymph node (SLN) biopsy is a common diagnostic strategy. SLNs have been studied using healthy rodents and large animals without metastasis. Here we used immunodeficient swine to establish a model of lymph node metastasis. We used RAG2-knockout immunodeficient swine. A431 human epithelial carcinoma cells expressing green fluorescent protein were injected subcutaneously into the posterior sides of the auricle, forelimb and hindlimb of knockout swine. Indigo carmine dye was injected subcutaneously 8 weeks after tumour cell transplantation. SLNs were extracted, observed using a stereoscopic fluorescence microscope and analysed histologically using haematoxylin and eosin staining, and immunohistochemistry. Lymphoid follicles were found in wild-type swine, and a few aggregated lymphocytes and immature lymphoid follicles were observed in knockout swine. Fluorescence in the lymph nodes indicated metastasis of tumour cells to the lymph nodes. Tumour cells replaced lymph node architectures, showed high-grade nuclear atypia and formed irregular tumour nests. Our model may be useful for the preclinical validation of diagnostic methods and minimally invasive treatment of metastatic cancer.

Magnetically Promoted Rapid Immunofluorescence Staining for Frozen Tissue Sections.

Current immunohistochemistry methods for diagnosing abnormal cells, such as cancer cells, require multiple steps and can be relatively slow compared with intraoperative frozen hematoxylin and eosin staining, and are therefore rarely used for intraoperative examination. Thus, there is a need for novel rapid detection methods. We previously demonstrated that functionalized fluorescent ferrite beads (FF beads) magnetically promoted rapid immunoreactions. The aim of this study was to improve the magnetically promoted rapid immunoreaction method using antibody-coated FF beads and a magnet subjected to a magnetic field. Using frozen sections of xenograft samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also determined the optimum magnetic force for the antibody reaction (from 7.79 × 10-15 N to 3.35 × 10-15 N) and washing (4.78 × 10-16 N), which are important steps in this technique. Furthermore, we stained paraffin-embedded tissue arrays and frozen sections of metastatic breast cancer lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the utility of this system in clinical specimens. Under optimal conditions, this ultra-rapid immunostaining method may provide an ancillary method for pathological diagnosis during surgery. (J Histochem Cytochem 58:XXX-XXX, 2010).

Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation.

TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs.