Transgenic Models to Study Angiogenesis and Lymphangiogenesis: An Historical Note.

Developments in transgenic technology: applications for medicine

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61) are prototypical members of the CCN family which also contains nephroblastoma overexpressed (NOV) and Wnt-induced secreted proteins-1, -2 and -3 (WISP-1, -2, -3). These proteins function as extracellular matrix (ECM)-associated signaling molecules that contain structural modules allowing them to bind directly with other moieties in the pericellular environment. Although multiple target cell types have been identified for CCN proteins, there is strong evidence supporting a role for CTGF and CYR61 in the regulation of endothelial cell function and angiogenesis. Both CTGF and CYR61 can promote endothelial cell growth, migration, adhesion and survival in vitro and at least some of these effects are mediated through cell surface integrins. Both proteins are transcriptionally activated in endothelial cells in response to basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), and endothelial cell proliferation and migration in vitro is reduced by antagonists of CTGF production or action. The expression pattern of CTGF and CYR61 in endothelial cells of vessels in situ supports a role for these molecules in normal endothelial homeostasis, as well as participating in the angiogenic process during embryonic development, placentation, tumor formation, fibrosis, and wound healing. CTGF or CYR61 knockout mice exhibit vascular defects during embryogenesis and fetal development. Both CTGF and CYR61 are intrinsically active in in vivo asssays for angiogenic activity. However, they can also regulate the production and/or activity of other angiogenic molecules (e.g. bFGF, VEGF) as well molecules that affect the integrity or stability of the ECM (e.g. collagen, matrix metalloproteases (MMPs), tissue inhibitors of MMPs (TIMPs)). Therefore, through their paracrine action as products of cells such as fibroblasts or smooth muscle cells or through their autocrine action as products of endothelial cells, CTGF and CYR61 participate in a variety of direct and indirect mechanisms by which angiogenesis is regulated at multiple control points.

Transgenic Mouse Models of Angiogenesis and Lymphangiogenesis

Technology development is seminal to many aspects of basic and applied plant transgenic science. Through the development and commercialization of genetically modified crops, the evolution of plant transgenic technologies is also relevant to society as a whole. In this study, literature statistics were used to uncover trends in the development of these technologies. Publication volume and impact (citation) over the past 30 years were analysed with respect to economic zones, countries, species and DNA delivery method. This revealed that, following a dramatic expansion in the 1980s, publications focusing on the development of transgenic technology have been slowing down worldwide since the early mid-1990s, except in a few leading Asian countries. The implications of these trends on the future of plant transgenic science as a whole are discussed.

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.

Introduction Animal models that recreate specific pathogenic events and their corresponding behavioral outcomes are indispensable tools for exploring the underlying pathophysiologic mechanisms of disease and for investigating therapeutic strategies prior to testing them in human patients. Although rodents have a long tradition as models for human neurological diseases, they have received increasing attention in light of genetic engineering methods that have made it possible to create precisely defined genetic changes. The development of transgenic technology, a tool that allows sophisticated manipulation of the genome, has provided an unprecedented opportunity to expand our understanding of many aspects of neuronal development, function, and disease. Transgenic animals are specific variants of species following the introduction and/or integration of a new gene or genes into the genome of the host animal. Transgenic technology is now routinely used to increase the level of (overexpress) particular proteins or enzymes in animals or to mutate or inactivate a particular gene using a “knockout” approach. One of the most commonly used animals in transgenic techniques is the mouse, a species in which transgene microinjections into the pronuclei of fertilized oocytes and the subsequent expression of the transgene in the animal have been carefully worked out. Since their development in the 1980s, the transgenic and knockout technologies have allowed us to examine the regulation of gene expression and the pathophysiology of its alterations.

The advantages of genetically modified food are gradually highlighted because of the rapid development of modern transgenic technology. The trade proportion of genetically modified food in international trade products is increasing. Till now, legal regulations concerning the international trade of genetically modified food are still contradictory and conflicting, and a series of initiated legal issues about the international trade of genetically modified food becomes more acute. As a great power of transgenic crops planting, China should fully learn from the trade disputes of genetically modified food, perfect laws such as the safety management, approval and identification of import and export, accelerate the development of transgenic technology, and strengthen the mutual benefit and collaboration of the developing countries, thus to gain a strong competitive position in international trade and maintain the fundamental interests of China better.

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.

Approaches to modeling stromal-epithelial interactions.

To explore the functional role of thrombospondin 2 (THBS2) in the metastasis of skin cutaneous melanoma (SKCM), with a focus on its regulation of angiogenesis and extracellular matrix (ECM) remodeling. THBS2 expression was assessed in normal melanocytes and SKCM cell lines with varying metastatic potential. Functional analyses were conducted after THBS2 knockdown in A375 cells and overexpression in G-361 cells. Effects on migration, invasion, endothelial tube formation, and angiogenesis- and ECM-related factors were evaluated. Tumor IMmune Estimation Resource database was used for correlation analyses in SKCM samples. A liver metastasis model was established by intrasplenic injection of B16-F10 cells into Thbs2 knockout and wild-type mice, followed by quantification of hepatic metastases and molecular analysis of peritumoral liver tissue. THBS2 was highly expressed in invasive melanoma cell lines and was positively associated with VEGFA, PECAM1, and MMPs in both databases and experimental models. Knockdown of THBS2 significantly suppressed VEGFA, PECAM1, FGF2, FLT1, MMP2, MMP9, and ECM components (LAMA4, COL1A1, and COL4A1) at mRNA and protein levels, inhibited melanoma cell migration and invasion, and reduced tube formation in human umbilical vein endothelial cells. Overexpression had opposite effects. In vivo , Thbs2 knockout mice exhibited significantly fewer hepatic metastases and reduced metastatic area compared with wild-type controls. Expression of Lama4, Pecam1, Vegfa, Mmp2, and Mmp9 was markedly lower in peritumoral liver tissue of knockout mice. THBS2 promotes SKCM metastasis by enhancing angiogenesis and ECM remodeling. Targeting THBS2 may represent a promising strategy for inhibiting melanoma progression and distant organ colonization.

BackgroundLipid synthesis is an indispensable process during embryo and growth development. Abnormal lipid synthesis metabolism can cause multiple metabolic diseases including obesity and hyperlipidemia. Stearoyl-Coenzyme A desaturase 1 (SCD1) is responsible for catalyzing the synthesis of monounsaturated fatty acids (MUFA) and plays an essential role in lipid metabolism. The aim of our study was to evaluate the effects of SCD1 on embryo development and lipid synthesis in a knockout mice model.MethodsWe used the CRISPR/Cas9 system together with microinjection for the knockout mouse model generation. Ten-week-old female C57BL/6 mice were used for zygote collection. RNase-free water was injected into mouse zygotes at different cell phases in order to select the optimal time for microinjection. Five sgRNAs were designed and in vitro transcription was performed to obtain sgRNAs and Cas9 mRNA. RNase-free water, NC sgRNA/Cas9 mRNA, and Scd1 sgRNA/Cas9 mRNA were injected into zygotes to observe the morula and blastocyst formation rates. Embryos that were injected with Scd1 sgRNA/Cas9 mRNA and developed to the two-cell stage were used for embryo transfer. Body weight, triacylglycerol (TAG), and cholesterol in Scd1 knockout mice serum were analyzed to determine the effects of SCD1 on lipid metabolism.ResultsMicroinjection performed during the S phase presented with the highest zygote survival rate (P < 0.05). Of the five sgRNAs targeted to Scd1, two sgRNAs with relatively higher gene editing efficiency were used for Scd1 knockout embryos and mice generation. Genome sequence modification was observed at Scd1 exons in embryos, and Scd1 knockout reduced blastocyst formation rates (P < 0.05). Three Scd1 monoallelic knockout mice were obtained. In mice, the protein level of SCD1 decreased (P < 0.05), and the body weight and serum TAG and cholesterol contents were all reduced (P < 0.01).

Global trends in plant transgenic science and technology (1973–2003)

With the development of transgenic technology, transgenic food is more and more common, which has attracted a great deal of controversy. This article expounded the quality of the food transgenic characteristics of meanings from the product analysis, and analyzed the quality of genetically modified food with the help of the existing economic theory. Genetically modified characteristics of food contain two levels. One type is genetically modified ingredients, influence of genetically modified ingredients for food use value, the society and environment. The other type is the presence of genetically modified ingredients in food, and levels of genetically modified ingredients. In the study of economics, the genetically modified features of food has important meaning, which was showed by the food genetically modified distribution and inspection of quality information, as well as the consumers' preference difference, cognitive and stability. Features of food Food features literally refers to the properties and characteristics of food, which is the performance in the raw material, appearance, processing and cooking. From the point of performance, features are the biological, physical and chemical indicators or parameters of the product. For food production, transgenic technology is a kind of new breeding techniques; it realized the biological properties of genetic modification without the traditional sexual breeding process through genetic recombination. Transgenic breeding process mainly includes three stages, the first is genetic screening and artificial separation of genes, and then build the genetic transformation of the carrier, the final is to import the purpose genes. Gm varieties obtained by transgenic technology enter into agricultural production, and was seen as raw materials for the food or for food processing. Because of orientation and accuracy of the transgenic technology, it has great potential in food production. At present, genetically modified (GM Traits) has been in commercial production is mainly herbicide resistance and resistance to pesticides. From existing types of food, genetically modified foods are generally has a corresponding traditional food, which are almost identical on existing food features, the most important difference is in genetically modified ingredients. That is to say, with the emergence of transgenic technology and transgenic food, genetically modified (gm) features have become food intrinsic features.

ADAMTS18 Metalloproteinase Regulates Angiogenesis In Vivo: Evidence from ADAMTS18 Deficient Mice and Zebrafish