Hatchability evaluation of bovine IVF embryos using OCT-based 3D image analysis.

Although embryo transfer is widely applied in cattle, many of the transferred embryos do not result in pregnancy. To determine a new parameter for bovine embryo evaluation, we investigated the relationships between in vitro hatchability and embryo morphological parameters using optical coherence tomography (OCT) that we established recently. Bovine embryos were obtained from Japanese Black cattle by in vitro fertilization (IVF). The quality of the blastocysts was examined under an inverted microscope and confirmed as Codes 1-3 according to the IETS standards for embryo evaluation. The OCT images of the embryos were captured on Day 7 after IVF, and the embryos were cultured until Day 9 to determine their hatchability. During OCT, the embryos were irradiated with near-infrared light for a few minutes to obtain three-dimensional images. In total, 22 parameters were assessed for each of the 42 embryos, of which 25 hatched (H embryos) and 17 did not (NH embryos). The thickness of the trophectoderm (TE) and TE+zona pellucida (ZP) was lesser, and the volumes of the TE, ZP, blastocoel, and whole embryo and blastocoel diameter were greater in the H embryos than in the NH embryos. PCA identified that the increase in the blastocoel-related value along with the decrease in the thickness-related value of the TE and/or ZP could be indicators for evaluating the hatchability of bovine IVF embryos. These results support the idea that OCT-captured structural data of blastocyst-stage embryos can be used as a potential model to predict the quality of bovine embryos.

Kidney organoid derived from renal tissue stem cells is a useful tool for histopathological assessment of nephrotoxicity in a cisplatin-induced acute renal tubular injury model.

Organoids derived from renal tissue stem cells (KS cells) isolated from the S3 segment of adult rat nephrons have previously been developed and evaluated. However, data regarding the histopathological evaluation of these organoids are limited. Therefore, in this study, we performed histopathological examinations of the properties of these organoids and evaluated the nephrotoxicity changes induced by cisplatin treatment. We observe that the tubular structure of the organoids was generally lined by a single layer of cells, in concordance with previous findings. Microvilli were exclusively observed under electron microscopy on the luminal side of this tubular structure. Moreover, the luminal side of the tubular structure was positive for aquaporin-1 (Aqp1), a marker of the proximal renal tubule. Cisplatin treatment induced cell death and degeneration, including cytoplasmic vacuolation, in cells within the tubular structure of the organoids. Cisplatin toxicity is associated with the induction of γ-H2AX (a marker of DNA damage) and the drop of phospho-histone H3 (a marker of cell division) levels. During the nephrotoxicity assessment, the kidney organoids displayed various features similar to those of the natural kidney, suggesting that it is possible to use these organoids in predicting nephrotoxicity. The histological evaluation of the organoids in this study provides insights into the mechanisms underlying nephrotoxicity.

Three-dimensional live imaging of bovine embryos by optical coherence tomography.

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.

Latent TGF-ß binding protein-2 is essential for the development of ciliary zonule microfibrils.

Latent TGF-ß-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

A kidney injury molecule-1 (Kim-1) gene reporter in a mouse artificial chromosome: the responsiveness to cisplatin toxicity in immortalized mouse kidney S3 cells.

BACKGROUND: Kidney injury molecule-1 (Kim-1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim-1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim-1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells, such as human kidney-2 cells and immortalized proximal tubular epithelial cells. METHODS: We measured the augmentation of Kim-1 mRNA expression after the addition of cisplatin using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature-sensitive large T antigen from Simian virus 40. RESULTS: A mouse Kim-1 gene luciferase reporter in conjunction with an Hprt gene reporter detected cisplatin-induced nephrotoxicity in S3 cells. These two reporter genes were contained in a mouse artificial chromosome, and two luciferases that emitted different wavelengths were used to monitor the respective gene expression. However, the Kim-1 reporter gene failed to respond to cisplatin in A9 fibroblast cells that contained the same reporter mouse artificial chromosome, suggesting cell type-specificity for activation of the reporter. CONCLUSIONS: We report the feasibility of measuring in vitro cisplatin nephrotoxicity using a Kim-1 reporter gene in S3 cells.

Exploring the origin and limitations of kidney regeneration.

Epidemiological findings indicate that acute kidney injury (AKI) increases the risk for chronic kidney disease (CKD), although the molecular mechanism remains unclear. Genetic fate mapping demonstrated that nephrons, functional units in the kidney, are repaired by surviving nephrons after AKI. However, the cell population that repairs damaged nephrons and their repair capacity limitations remain controversial. To answer these questions, we generated a new transgenic mouse strain in which mature proximal tubules, the segment predominantly damaged during AKI, could be genetically labelled at desired time points. Using this strain, massive proliferation of mature proximal tubules is observed during repair, with no dilution of the genetic label after the repair process, demonstrating that proximal tubules are repaired mainly by their own proliferation. Furthermore, acute tubular injury caused significant shortening of proximal tubules associated with interstitial fibrosis, suggesting that proximal tubules have a limited capacity to repair. Understanding the mechanism of this limitation might clarify the mechanism of the AKI-to-CKD continuum.

Repression of cyclin D1 expression is necessary for the maintenance of cell cycle exit in adult mammalian cardiomyocytes.

The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.

Exploring new gene integration sites for gene knock-in by gene-trapping strategy.

The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Rosa26 locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent-positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.

Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.

The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10(-6)). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.

Three-dimensional morphology of bovine blastocysts hatched against lipopolysaccharide exposure in vitro.

Embryo transfer in cattle is globally becoming more ubiquitous, but the pregnancy rate is lower than that of artificial insemination. The uterus contains its own bacteria, and concentrations of lipopolysaccharides (LPS) from gram-negative bacteria are higher in uteri affected by endometritis than in healthy uteri and they suppress embryogenesis. The purpose of this study was to investigate the morphological characteristics of bovine embryos with a higher viability and implantability, by analyzing the morphology of bovine blastocysts that successfully hatched under challenge of LPS, using an optical coherence tomography (OCT) system. Developing embryos produced by in vitro fertilization that had reached the blastocyst stage on Day 7 were three-dimensionally scanned using an OCT system, then were continued to culture with or without LPS until Day 9, when the presence or absence of hatching was determined. The OCT-captured three-dimensional images were used to quantify 20 different metrics, including inner cell mass (ICM), trophectoderm, blastocoel, and total embryo volume; each of the parameters was compared between the hatched and unhatched embryos. Under the LPS challenge, hatched embryos had higher ICM thickness and volume, and lower trophectoderm thickness than unhatched embryos. Furthermore, hatched embryos under LPS challenge had higher ICM thickness and ICM volume than hatched embryos without LPS challenge. The present results suggest the possibility that ICM thickness and ICM volume calculated by OCT system could be indices for good quality bovine embryos.

Cisplatin Induces Cell Death in Rat Adult Kidney Stem/ Progenitor Cell-Derived Kidney Organoids.

Background: The use of stem/ progenitor cell-derived organoids to evaluate the toxicity of chemical substances has widely increased. Organoids with nephron-like structures (NLS) can be derived from rat adult kidney stem/ progenitor cells (rKS cells) using three-dimensional culture. In this study, we examined the effects of cisplatin, an anticancer drug that induces nephrotoxicity in vivo, on rKS cell-derived NLS. Methods: Twelve organoids were simultaneously derived from three-dimensionally cultured rKS cells in Matrigel matrices. The surface area of each organoid was measured using microscopy-based imaging, and the morphological changes of NLS were monitored using an image analysis method. NLS were exposed to cisplatin, and their associated effects were assessed. Results: NLS elongated over time. The surface areas of the 12 organoids were almost constant. Cisplatin exposure induced cell death in NLS and inhibited the increase in the surface area of the organoids. Conclusion: Cisplatin exposure induces damage to NLS derived from rKS cells. Thus, the organoids may be valuable as an in vitro model to assess nephrotoxicity.

Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo.

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat-containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5-deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171-175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168-171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.

Fibulin-4 conducts proper elastogenesis via interaction with cross-linking enzyme lysyl oxidase.

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.

Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation.

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

The Influence of Illumination Color on the Subjective Visual Recognition of Biological Specimens.

BACKGROUND: Visual examination by the naked eye is integral to medical diagnosis and surgery. The illumination in conditioned color is widely used for visual inspection in the industry but has not been introduced to the biomedical context. The color that can enhance the visual recognition of individual tissues is still unknown. Therefore, we carried out a visual recognition experiment on biological specimens to determine the subjective preference for illumination color based on questionnaires. METHODS: Twenty healthy subjects were asked to compare the visual recognizability of several rat tissues between the illuminations in test colors and white. The rats were anesthetized, and the femoral vein and abdominal cavity were exposed. Seven tissues were selected for a visual recognition test. Illumination was generated using a multi-color LED light. The subjects observed the tissues under the illuminations of white and one of the test colors alternately and reported which illumination is suitable for visual recognition using a questionnaire. RESULTS: The analysis of the questionnaires showed that the blue test color was more effective than white illumination in the visual recognition of fine structures such as the branching of blood vessels and nerves, and red illumination disturbed the visual recognizability of the same tissues. On the other hand, the red but not the blue illumination improved the visual recognizability of the vein beneath the intact skin. As to the recognition of individual tissues in the abdominal cavity, the white illumination gave a better visual recognizability compared to every other test color. CONCLUSION: This study shows that the illumination color influences the visual recognition of biological specimens and the adequate color for the visual recognition of specific tissue parts is distinct among biological specimens. Using the lighting system to make fine adjustments to the illumination color may be useful in medical diagnosis and surgery.

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation.

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.

New insights into the efficacy of SR-16234, a selective estrogen receptor modulator, on the growth of murine endometriosis-like lesions.

PROBLEM: To evaluate the effects of SR-16234 (SR), a selective estrogen receptor modulator (SERM), on murine endometriosis-like lesions. METHOD OF STUDY: BALB/c mice (n = 53) were used to establish the murine endometriosis model. Ovariectomized, estradiol replaced, 6-week-old murine endometriosis model were injected with lipopolysaccharide (LPS) with or without SR (1 mg/kg/d) or vehicle, over a period of 4 weeks. Upon treatment completion, the endometriosis-like lesions that developed in the abdominal cavity of mice were counted, measured, and collected. Gene expression of inflammatory cytokines and estrogen receptor (ER) in the lesions was assessed by real-time RT-PCR. Immunohistochemical analysis was used to evaluate the effect of SR on cell proliferation, angiogenic activity, inflammation, and NF-κB phosphorylation. RESULTS: Treatment with SR significantly reduced the total number and size of lesions per mouse without inducing endometrial growth. In addition, SR downregulated LPS-enhanced Vegf, Il-6, Ptgs-2, and Ccl-2 and ER mRNA expression in endometriosis-like lesions. Immunohistochemical analysis demonstrated a decrease in percentage of positive cells of Ki67, and intensity and rate of positive cells of ERα, CD3, F4/80, PECAM by SR treatment. SR also decreased the expression of NF-κB p65 and phospho-NF-κB p65. CONCLUSION: SR has a regressive effect on the development of murine endometriosis-like lesions.

Inhibition of IAP (inhibitor of apoptosis) proteins represses inflammatory status via nuclear factor-kappa B pathway in murine endometriosis lesions.

PROBLEM: How is the role of inhibitor of apoptosis proteins (IAPs) in the development of murine endometriosis lesions? METHOD OF STUDY: BALB/c female mice (n = 36) were used for the murine endometriosis model. Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue. After 4 weeks of IAP antagonist (BV6) treatment, the expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Inflammatory state, angiogenic activity, and nuclear factor-kappa B (NF-κB) activation were assessed by immunohistochemical staining. RESULTS: The number, size, and level of inflammatory cytokines (Vegf, Il-6, Ccl-2, Lif) gene expression in the murine endometriosis-like lesions were reduced by BV6 treatment. BV6 repressed the intensity and rate of positive cells of CD3, F4/80, and PECAM immunostaining; in addition, the expression of NF-κB p65 and phospho-NF-κB p65 was also attenuated. CONCLUSION: Inhibitor of apoptosis proteins antagonist represses the inflammation status of murine endometriosis-like lesions viaNF-κB pathway. IAPs may be a novel therapeutic target for endometriosis.

Development of a multiple-gene-loading method by combining multi-integration system-equipped mouse artificial chromosome vector and CRISPR-Cas9.

Mouse artificial chromosome (MAC) vectors have several advantages as gene delivery vectors, such as stable and independent maintenance in host cells without integration, transferability from donor cells to recipient cells via microcell-mediated chromosome transfer (MMCT), and the potential for loading a megabase-sized DNA fragment. Previously, a MAC containing a multi-integrase platform (MI-MAC) was developed to facilitate the transfer of multiple genes into desired cells. Although the MI system can theoretically hold five gene-loading vectors (GLVs), there are a limited number of drugs available for the selection of multiple-GLV integration. To overcome this issue, we attempted to knock out and reuse drug resistance genes (DRGs) using the CRISPR-Cas9 system. In this study, we developed new methods for multiple-GLV integration. As a proof of concept, we introduced five GLVs in the MI-MAC by these methods, in which each GLV contained a gene encoding a fluorescent or luminescent protein (EGFP, mCherry, BFP, Eluc, and Cluc). Genes of interest (GOI) on the MI-MAC were expressed stably and functionally without silencing in the host cells. Furthermore, the MI-MAC carrying five GLVs was transferred to other cells by MMCT, and the resultant recipient cells exhibited all five fluorescence/luminescence signals. Thus, the MI-MAC was successfully used as a multiple-GLV integration vector using the CRISPR-Cas9 system. The MI-MAC employing these methods may resolve bottlenecks in developing multiple-gene humanized models, multiple-gene monitoring models, disease models, reprogramming, and inducible gene expression systems.