Transgenic Model Organisms Research Articles

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

In Vitro Fertilization of the African Turquoise Killifish Nothobranchius furzeri.

The ability to perform in vitro fertilization, together with sperm cryopreservation, greatly facilitates the long-term laboratory maintenance of wild-type and transgenic model organisms and helps prevent genetic drift. It is also useful in cases where reproduction may be compromised. In this protocol, we present a method for in vitro fertilization of the African Turquoise killifish Nothobranchius furzeri that is compatible with the use of fresh or cryopreserved sperm.

Pleiotropic Actions of PGRMC Proteins in Cancer.

Progesterone receptor membrane component (PGRMC) proteins play important roles in tumor growth, progression, and chemoresistance, of which PGRMC1 is the best characterized. The ancestral member predates the evolution of metazoans, so it is perhaps not surprising that many of the purported actions of PGRMC proteins are rooted in fundamental metabolic processes such as proliferation, apoptosis, and DNA damage responses. Despite mediating some of the actions of progesterone (P4) and being fundamentally required for female fertility, PGRMC1 and PGRMC2 are broadly expressed in most tissues. As such, these proteins likely have both progesterone-dependent and progesterone-independent functions. It has been proposed that PGRMC1 acquired the ability to mediate P4 actions over evolutionary time through acquisition of its cytochrome b5-like heme/sterol-binding domain. Diverse reproductive and nonreproductive diseases associate with altered PGRMC1 expression, epigenetic regulation, or gene silencing mechanisms, some of which include polycystic ovarian disease, premature ovarian insufficiency, endometriosis, Alzheimer disease, and cancer. Although many studies have been completed using transformed cell lines in culture or in xenograft tumor approaches, recently developed transgenic model organisms are offering new insights in the physiological actions of PGRMC proteins, as well as pathophysiological and oncogenic consequences when PGRMC expression is altered. The purpose of this mini-review is to provide an overview of PGRMC proteins in cancer and to offer discussion of where this field must go to solidify PGRMC proteins as central contributors to the oncogenic process.

Simple Recommendations for Improving Efficiency in Generating Genome-Edited Mice.

The generation of transgenic model organisms (primarily mice) is an integral part of modern fundamental and applied research. Simple techniques based on the biology of these laboratory rodents can often increase efficiency when generating genome-edited mouse strains. In this study, we share our three years of experience in the optimization of mouse genome editing based on microinjection of CRISPR/Cas9 components into ca. 10,000 zygotes. We tested a number of techniques meant to improve efficiency in generating knockout mice, such as optimization of the superovulation method and choosing the optimal mouse strains to be used as zygote donors and foster mothers. The presented results might be useful to laboratories aiming to quickly and efficiently create new mouse strains with tailored genome editing.

Simplified platform for mosaic in vivo analysis of cellular maturation in the developing heart

Cardiac cells develop within an elaborate electro-mechanical syncytium that continuously generates and reacts to biophysical force. The complexity of the cellular interactions, hemodynamic stresses, and electrical circuitry within the forming heart present significant challenges for mechanistic research into the cellular dynamics of cardiomyocyte maturation. Simply stated, it is prohibitively difficult to replicate the native electro-mechanical cardiac microenvironment in tissue culture systems favorable to high-resolution cellular/subcellular analysis, and current transgenic models of higher vertebrate heart development are limited in their ability to manipulate and assay the behavior of individual cells. As such, cardiac research currently lacks a simple experimental platform for real-time evaluation of cellular function under conditions that replicate native development. Here we report the design and validation of a rapid, low-cost system for stable in vivo somatic transgenesis that allows for individual cells to be genetically manipulated, tracked, and examined at subcellular resolution within the forming four-chambered heart. This experimental platform has several advantages over current technologies, chief among these being that mosaic cellular perturbations can be conducted without globally altering cardiac function. Consequently, direct analysis of cellular behavior can be interrogated in the absence of the organ level adaptions that often confound data interpretation in germline transgenic model organisms.

Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.

Chronic low‐dose radiation inhibits cisplatin‐induced formation of tumorous clones in Drosophila melanogaster wts/ + heterozygotes

AbstractIonizing radiation is one of the most extensively studied carcinogens. In contrast to the detrimental effects of high‐dose radiation in carcinogenesis, the biological effects of low‐dose radiation remains poorly understood. In this study, we introduced adult wts/ + heterozygotes of Drosophila melanogaster as transgenic model organisms to determine the tumorigenic activity of low‐dose radiation. The warts (wts) gene is a tumor suppressor gene in mice and humans that is directly involved in cell cycle regulation. Fruit flies at the first larval stage were subjected to ionizing radiation, and then tumorigenic activity was evaluated as the frequency of observed warts tumorous mosaic clones in adult flies. Low‐dose irradiation alone did not cause tumorigenesis in our system. In combined treatment with a chemical carcinogen, chronic irradiation at 0.2 Gy decreased the frequency of tumorous clones induced by cisplatin. These results suggest that low‐dose radiation alone is not deleterious but beneficial in tumorigenesis induced by a chemical carcinogen.

Transgenic Epigenetics: Using Transgenic Organisms to Examine Epigenetic Phenomena

Non-model organisms are generally more difficult and/or time consuming to work with than model organisms. In addition, epigenetic analysis of model organisms is facilitated by well-established protocols, and commercially-available reagents and kits that may not be available for, or previously tested on, non-model organisms. Given the evolutionary conservation and widespread nature of many epigenetic mechanisms, a powerful method to analyze epigenetic phenomena from non-model organisms would be to use transgenic model organisms containing an epigenetic region of interest from the non-model. Interestingly, while transgenic Drosophila and mice have provided significant insight into the molecular mechanisms and evolutionary conservation of the epigenetic processes that target epigenetic control regions in other model organisms, this method has so far been under-exploited for non-model organism epigenetic analysis. This paper details several experiments that have examined the epigenetic processes of genomic imprinting and paramutation, by transferring an epigenetic control region from one model organism to another. These cross-species experiments demonstrate that valuable insight into both the molecular mechanisms and evolutionary conservation of epigenetic processes may be obtained via transgenic experiments, which can then be used to guide further investigations and experiments in the species of interest.

Fluorescent Protein-Based Redox Probes

Redox biochemistry is increasingly recognized as an integral component of cellular signal processing and cell fate decision making. Unfortunately, our capabilities to observe and measure clearly defined redox processes in the natural context of living cells, tissues, or organisms are woefully limited. The most advanced and promising tools for specific, quantitative, dynamic and compartment-specific observations are genetically encoded redox probes derived from green fluorescent protein (GFP). Within only few years from their initial introduction, redox-sensitive yellow FP (rxYFP), redox-sensitive GFPs (roGFPs), and HyPer have generated enormous interest in applying these novel tools to monitor dynamic redox changes in vivo. As genetically encoded probes, these biosensors can be specifically targeted to different subcellular locations. A critical advantage of roGFPs and HyPer is their ratiometric fluorogenic behavior. Moreover, the probe scaffold of redox-sensitive fluorescent proteins (rxYFP and roGFPs) is amenable to molecular engineering, offering fascinating prospects for further developments. In particular, the engineering of redox relays between roGFPs and redox enzymes allows control of probe specificity and enhancement of sensitivity. Genetically encoded redox probes enable the functional analysis of individual proteins in cellular redox homeostasis. In addition, redox biosensor transgenic model organisms offer extended opportunities for dynamic in vivo imaging of redox processes.

Implementation and enforcement of the 3Rs principle in the field of transgenic animals used for scientific purposes

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals (Fig. 1). These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or threedimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified (“humanised”) animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with “negative” outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use.The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.

Improved behavioral response as a valid biomarker for drug screening program in transgenic rodent models of tauopathies.

Neurodegenerative tauopathies are defined as a group of dementia and movement disorders characterized by prominent filamentous tau inclusions and degeneration located within certain brain regions. Their common sign is a presence of proteinaceous aggregates composed of hyperphosphorylated and truncated tau proteins. The molecular mechanisms of the disease still remain unresolved, therefore transgenic organisms displaying tau-related neurodegenerative cascade have been created to allow decoding of individual pathways involved in human pathological conditions. Moreover, use of transgenic model organisms enables the application of potential therapeutic approaches. The expression of mutated or misfolded tau as a transgene in vivo leads to significant alteration of neurobehavioral features of experimental animal, therefore detailed classification of behavioral phenotype become one of the first crucial analyses, while it functionally correlates with central nervous system impairment. Currently, two major types of behavioral impairment have been described in transgenic rodent models of tauopathies, (1) progressive motor impairment associated with muscular weakness and premature death and (2) age-related impairment of cognitive functions attended with unaffected motor status. Up to the present, only transgenic models displaying motor impairment were successfully applied into the drug trials targeting misfolded tau protein, despite their behavioral inconsistence with clinical profile of progressive human tauopathy. The aim of this study was, therefore, to summarize the pros and cons of used transgenic rodent models mimicking human tauopathies in connection with development of therapeutic strategies.

Age-related Cardiac Deterioration: insights from Drosophila

As the average lifespan in Western countries continues to expand, health care for the aged has become an increasingly important research focus. While clinicians and vertebrate researchers have frequently concentrated on specific age-related diseases, particularly neurodegenerative diseases such as Alzheimer's and Parkinson's Diseases, researchers working with invertebrate genetic model systems have gained important insights into global mechanisms of lifespan determination. Still others have employed biochemical and molecular approaches to elucidate processes contributing to common diseases of the elderly, such as cancer and diabetes. In between the broad focus on organismal aging and the more narrow focus on cellular dysfunction is the study of aging at the level of individual organ function. This review will attempt to highlight recent advances in the area of age-related deterioration of organ function provided by the use of transgenic model organisms, with a view toward incorporating these observations into a framework provided by both broader theories of the aging process and studies of cellular function during aging.

Mechanisms and models of α-synuclein-related neurodegeneration

Expression of the Parkinson's disease-associated protein alpha-synuclein causes formation of aggregates and cytotoxicity in a great diversity of transgenic model organisms, in the case of Drosophila melanogaster affecting specific dopaminergic neuron clusters. The relative contribution of alpha-synuclein misfolding and phosphorylation for neurodegeneration was elucidated in these systems. In transgenic mice, typical neuropathologic inclusions formed concomitant with behavioral deficits, reminiscent of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Neuronal degeneration was cell-autonomous in the Lewy body disease models, whereas gliotic changes accompanied neurodegeneration caused by (oligodendro)glial cytoplasmic inclusions. These recent findings provided major insights into the molecular mechanisms of alpha-synucleinopathies.