Construction of fluorescent transgenic zebrafish Tg (ttn.2: EGFP) / 生物工程学报

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.

Therapeutic potential of targeting SIRT1 for the treatment of Alzheimer's disease / 生理学报

Silent information regulator 1 (SIRT1) is one of the seven mammalian proteins of the sirtuin family of NAD+-dependent deacetylases. SIRT1 plays a pivotal role in neuroprotection and ongoing research has uncovered a mechanism by which SIRT1 may exert a neuroprotective effect on Alzheimer's disease (AD). Growing evidence demonstrates that SIRT1 regulates many pathological processes including amyloid-β precursor protein (APP) processing, neuroinflammation, neurodegeneration, and mitochondrial dysfunction. SIRT1 has recently received enormous attention, and pharmacological or transgenic approaches to activate the sirtuin pathway have shown promising results in the experimental models of AD. In the present review, we delineate the role of SIRT1 in AD from a disease-centered perspective and provides an up-to-date overview of the SIRT1 modulators and their potential as effective therapeutics in AD.

The in vivo imaging of zebrafish for chemical vascular toxicity assessment / 中华预防医学杂志

Objective: To evaluate the vascular toxicity of chemicals by a real-time observation approach using the transgenic zebrafish. Methods: The spatiotemporal vascular alterations of transgenic zebrafish after chemical exposure were assessed by laser confocal microscopy and high-content screening analysis, respectively. Results: The method using Laser Confocal Microscopy (LCM) is easier to operate and yields high-resolution images, while it is lower throughput and inefficient. In contrast, high-content analysis (HCA) analysis obtains high-quality data of vascular toxicity manifesting whole blood vasculature, whereas it requires delicate operation procedures and advanced experimental conditions. Conclusion: Two kinds of zebrafish imaging methods each have advantages and disadvantages. LCM is suitable for the evaluation of a small number of chemicals. HCA, a cutting-edge technology, has great potential for chemical safety assessment allowing high throughput vascular toxicity tests of a good number of chemicals at a time.

Functional characterization of piggyBac-like elements from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

PiggyBac is a transposable DNA element originally discovered in the cabbage looper moth (Trichoplusia ni). The T. ni piggyBac transposon can introduce exogenous fragments into a genome, constructing a transgenic organism. Nevertheless, the comprehensive analysis of endogenous piggyBac-like elements (PLEs) is important before using piggyBac, because they may influence the genetic stability of transgenic lines. Herein, we conducted a genome-wide analysis of PLEs in the brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), and identified a total of 28 PLE sequences. All N. lugens piggyBac-like elements (NlPLEs) were present as multiple copies in the genome of BPH. Among the identified NlPLEs, NlPLE25 had the highest copy number and it was distributed on five chromosomes. The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals, as well as a single open reading frame transposase encoding 546 amino acids. Furthermore, NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision. A cross-recognition between the NlPLE25 transposon and the piggyBac transposon was also revealed in this study. These findings provide useful information for the construction of transgenic insect lines.

Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system / 生理学报

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5 / 生物工程学报

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Mvda is required for zebrafish early development

BACKGROUND: The MVD gene mutations are identified in porokeratosis, which is considered a skin-specific autoin- flammatory keratinization disease. However, the biological function of MVD gene remains largely unknown. Therefore, we analyzed the function of mvda gene, orthologous to the human MVD gene, in developing zebrafish. METHODS: Morpholino antisense oligonucleotide technique was used to generate mvda loss-of-function phenotypes. Knockdown of mvda was confirmed by RT-PCR and Sanger sequencing. Scanning and transmission electron microscopy were performed to analyze the morphology of the epidermis. Angiogenesis study was presented using the Tg(fli1a:EGFP)yl transgenic strain. In addition, acridine orange staining was used to examine the apoptotic cells in vivo. RESULTS: As expected, the mvda morphants showed abnormal morphology of the epidermis. Moreover, we observed ectopic sprouts in trunk angiogenesis and impaired formation of the caudal vein plexus in the mvda-deficient zebrafish. Besides, increased apoptosis was found throughout the tail, heart, and eyes in mvda zebrafish morphants. CONCLUSIONS: These findings indicated the essential role of mvda in the early development of zebrafish. This was the first in vivo knockdown study of the zebrafish mvda gene, which might offer insight into the biological function of the human MVD gene.

Preparation of transgenic Musca domestica by microinjection method / 生物工程学报

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.

A double fluorescence chimeric limb regeneration model reveals muscle fiber reconnection during axolotl limb regeneration / Modelo de regeneración quimérica de doble fluorescencia del miembro revela la reconexión de la fibra muscular durante la regeneración del miembro axolotl

SUMMARY: Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages.

RESUMEN: La regeneración del miembro de Axolotl es una característica fascinante que ha llamado la atención durante varias décadas. Nuestros estudios previos sobre la regeneración del miembro del Axolotl indicaron que las células satélite en los músculos remanentes se mueven distalmente hacia el blastema para regenerar nuevos músculos que están separados por una brecha de músculos remanentes. A partir de entonces, las fibras musculares regenerativas comienzan a reconectarse con las restantes. En este estudio, se aclaró la reconexión a nivel de fibra muscular individual para probar la hipótesis de que esta reconexión ocurre sincrónicamente entre los músculos involucrados. Se trasplantaron tres pares de blastemas EGFP+ en la etapa de yema media en tocones recién amputados de axolotls RFP+ en la misma posición del muslo para generar miembros posteriores regenerativos quiméricos de fluorescencia doble. Estos miembros regenerativos se cosecharon muy tarde mucho más allá de haber alcanzado la etapa de diferenciación tardía. Las imágenes de fluorescencia de estos miembros en secciones transversales revelaron que en la parte remanente proximal de la fibra muscular, la reconexión se produjo a un ritmo diferente entre los músculos. En el músculo grácil, la reconexión comenzó desde la periferia antes de completarse. Además, las fibras musculares RFP+ contribuyeron a la regeneración muscular en las partes regenerativas distales. Curiosamente, esta contribución de glóbulos rojos se limitó a los músculos superficiales ventrales de la pantorrilla. Este tipo de modelo de regeneración quimérica de doble fluorescencia del miembro puede ayudar a aumentar la comprensión del patrón de la regeneración del miembro del Axolotl en etapas tardías.

Animal models of human atherosclerosis: current progress

Atherosclerosis retains the leading position among the causes of global morbidity and mortality worldwide, especially in the industrialized countries. Despite the continuing efforts to investigate disease pathogenesis and find the potential points of effective therapeutic intervention, our understanding of atherosclerosis mechanisms remains limited. This is partly due to the multifactorial nature of the disease pathogenesis, when several factors so different as altered lipid metabolism, increased oxidative stress, and chronic inflammation act together leading to the formation and progression of atherosclerotic plaques. Adequate animal models are currently indispensable for studying these processes and searching for novel therapies. Animal models based on rodents, such as mice and rats, and rabbits represent important tools for studying atherosclerosis. Currently, genetically modified animals allow for previously unknown possibilities in modelling the disease and its most relevant aspects. In this review, we describe the recent progress made in creating such models and discuss the most important findings obtained with them to date.

An update on the tools for creating transgenic animal models of human diseases - focus on atherosclerosis

Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.

Increasing transgenic expression in recombinant Chinese hamster ovary cells using introns in different directions / 生物工程学报

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.

Polydeoxyribonucleotides Improve Diabetic Wound Healing in Mouse Animal Model for Experimental Validation

BACKGROUND: Wound healing mechanisms is believed to have effects similar to wound healing disorders in diabetic patients, including abnormal inflammatory cells, angiogenesis disorders, and reduced collagen synthesis. Therefore, reestablishment of structural and promoted angiogenesis could be beneficial to promote wound healing process. OBJECTIVE: Therefore, we investigated whether the polydeoxyribonucleotide (PDRN) that was self-production in Korea, could be useful as an intradermal injection for promoting wound healing. Also, we validate for wound healing effect of PDRN using healing-impaired (db/db) mice. METHODS: In this study, we confirmed the effects of PDRN by creating wound models in in vitro and in vivo model. Using an in vitro wound healing assay, we observed that PDRN stimulated closure of wounded monolayers of human fibroblast cells. PDRN (8.25 mg/ml) or phosphate-buffered saline (0.9% NaCl) was injected once daily into the dermis adjacent to the wound for 12 days after skin injury. RESULTS: Time course observations revealed that mice treated with PDRN showed accelerated wound closure and epidermal and dermal regeneration, enhanced angiogenesis. The wound area and depth decreased at 3, 6, 9, and 12 days after skin injury. Histological evaluation showed an increase of vascular endothelial growth factor, CD31, and collagen fibers in the PDRN group compared with the control group, indicating that PDRN was effective in the treatment of delayed wound healing caused by diabetes. CONCLUSION: This study suggests that our PDRN has a wound healing effect in transgenic animal models with cells and diabetes through angiogenesis.

Animal models for colorectal cancer / Modelos animais de carcinogênese colorretal

ABSTRACT Introduction: Colorectal cancer is a very frequent sort of neoplasm among the population, with a high mortality rate. It develops from an association of genetic and environmental factors, and it is related to multiple cell signaling pathways. Cell cultures and animal models are used in research to reproduce the process of disease development in humans. Of the existing animal models, the most commonly used are animals with tumors induced by chemical agents and genetically modified animals. Objective: To present and synthesize the main animal models of colorectal carcinogenesis used in the research, comparing its advantages and disadvantages. Method: This literature review was performed through the search for scientific articles over the last 18 years in PubMed and Science Direct databases, by using keywords such as "animal models", "colorectal carcinogenesis" and "tumor induction". Results: 1,2-dimethylhydrazine and azoxymethane are carcinogenic agents with high specificity for the small and large intestine regions. Therefore, the two substances are widely used. Concerning the genetically modified animal models, there is a larger number of studies concerning mutations of the APC, p53 and K-ras genes. Animals with the APC gene mutation develop colorectal neoplasms, whereas animals with p53 and K-ras genes mutations are able to potentiate the effects of the APC gene mutation as well as the chemical inducers. Conclusion: Each animal model has advantages and disadvantages, and some are individually efficient as to the induction of carcinogenesis, and in other cases the association of two forms of induction is the best way to obtain representative results of carcinogenesis in humans.

RESUMO Introdução: O câncer de cólon e reto é bastante frequente na população e com elevado índice de mortalidade. Ele se desenvolve a partir da associação de fatores genéticos e ambientais e está relacionado a múltiplas vias de sinalização celular. Para o estudo da doença são utilizados cultivos celulares e modelos animais, que sejam capazes de reproduzir o processo de desenvolvimento da doença em humanos. Dos modelos existentes, os mais comumente utilizados são os animais induzidos ao desenvolvimento tumoral por agentes químicos e os animais geneticamente modificados. Objetivo: Apresentar e sintetizar os principais modelos animais de carcinogênese colorretal utilizados na pesquisa, comparando suas vantagens e desvantagens. Método: Para o desenvolvimento dessa revisão foi realizada uma busca por artigos científicos dos últimos 18 anos nas bases de dados PubMed e Science Direct, utilizando como palavras-chave "modelos animais", "carcinogênese colorretal" e "indução tumoral". Resultado: O 1,2 dimetilhidrazina e o azoximetano são agentes carcinógenos com alta especificidade para o intestino delgado e grosso. Por isso, as duas substâncias são amplamente utilizadas. Dos modelos animais geneticamente modificados observa-se maior quantidade de estudos referentes às mutações dos genes APC, p53eK-ras. Os animais com mutação do gene APC desenvolvem neoplasias colorretais, enquanto que animais com mutações dos genes p53 e K-ras são capazes de potencializar os efeitos da mutação do gene APC, bem como dos indutores químicos. Conclusão: Cada modelo animal apresenta vantagens e desvantagens, sendo que alguns são individualmente eficientes na indução da carcinogênese, e em outros casos a associação de duas formas de indução é a melhor maneira de se obter resultados representativos da carcinogênese em humanos.

Taurine Transporter dEAAT2 is Required for Auditory Transduction in Drosophila / 神经科学通报·英文版

Drosophila dEAAT2, a member of the excitatory amino-acid transporter (EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. Here, we report that dEAAT2 is required for the larval startle response to sound stimuli. dEAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound. More importantly, expressing dEAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together, these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.

Hierarchical Control of Drosophila Sleep, Courtship, and Feeding Behaviors by Male-Specific P1 Neurons / 神经科学通报·英文版

Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. Here, we investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. We found that mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.

Paeoniflorin Promotes Angiogenesis in A Vascular Insufficiency Model of Zebrafish in vivo and in Human Umbilical Vein Endothelial Cells in vitro / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the pro-angiogenic effects of paeoniflorin (PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>In vivo, the pro-angiogenic effects of PF were tested in a vascular insufficiency model in the Tg(fli-1:EGFP)y1 transgenic zebrafish. The 24 h post fertilization (hpf) embryos were pretreated with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor II (VRI) for 3 h to establish the vascular insufficiency model and then post-treated with PF for 24 h. The formation of intersegmental vessels (ISVs) was observed with a fluorescence microscope. The mRNA expression of fms-like tyrosine kinase-1 (flt-1), kinase insert domain receptor (kdr), kinase insert domain receptor like (kdrl) and von Willebrand factor (vWF) were analyzed by real-time polymerase chain reaction (PCR). In vitro, the pro-angiogenic effects of PF were observed in HUVECs in which cell proliferation, migration and tube formation were assessed.</p><p><b>RESULTS</b>PF (6.25-100 μmol/L) could rescue VRI-induced blood vessel loss in zebrafish and PF (25-100 μmol/L), thereby restoring the mRNA expressions of flt-1, kdr, kdrl and vWF, which were down-regulated by VRI treatment. In addition, PF (0.001-0.03 μmol/L) could promote the proliferation of HUVECs while PF stimulated HUVECs migration at 1.0-10 μmol/L and tube formation at 0.3 μmol/L.</p><p><b>CONCLUSION</b>PF could promote angiogenesis in a vascular insufficiency model of zebrafish in vivo and in HUVECs in vitro.</p>

Production of transgenic pigs using a pGFAP-CreER(T2)/EGFP(LoxP) inducible system for central nervous system disease models

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreER(T2)/LCMV-EGFP(LoxP) and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreER(T2)) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreER(T2)-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER(T2)/EGFP(LoxP) recombination system via SCNT.

Role of cytochrome P450 epoxygenase-dependent arachidonic acid metabolites in kidney physiology and diseases / 生理学报

Kidney diseases are important causes of mortality world widely. Renal microvascular dysfunction plays a pivotal role in the development of kidney diseases. Pharmacological and biochemical tools have been used to conduct detailed studies on the metabolization of arachidonic acid by cytochrome P450 (CYP450) in renal microvasculature. CYP450 epoxygenase metabolites epoxyeicosatrienoic acids (EETs) are mainly produced in renal microvessels. EETs exhibit renoprotective effects through vasodilation, anti-hypertension, anti-apoptosis and anti-inflammation, and were reported as therapeutic targets of renal diseases. However, the ability of the kidney in generating EETs is reduced in renal diseases. Recently, the studies from transgenic animal overexpressing CYP450 epoxygenases and application of soluble epoxide hydrolase inhibitors revealed that increasing of EETs exhibits renoprotective effects in vivo. The present review focuses on the protective mechanisms of EETs in kidney physiology and diseases.