Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.     

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology¹. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.     

Label-Free Imaging of Lipid Depositions in C. elegans Using Third-Harmonic Generation Microscopy

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.     

Using C. elegans for aging research

Over a century ago, the zoologist Emile Maupas first identified the nematode, Rhabditis elegans, in the soil in Algiers. Subsequent work and phylogenic studies renamed the species Caenorhabditis elegans or more commonly referred to as C. elegans; (Caeno meaning recent; rhabditis meaning rod; elegans meaning nice). However, it was not until 1963, when Sydney Brenner, already successful from his work on DNA, RNA, and the genetic code, suggested the future of biological research lay in model organisms. Brenner believed that biological research required a model system that could grow in vast quantities in the lab, were cheap to maintain and had a simple body plan, and he chose the nematode C. elegans to fulfill such a role. Since that time, C. elegans has emerged as one of the premiere model systems for aging research. This paper reviews some initial identification of mutants with altered lifespan with a focus on genetics and then discusses advantages and disadvantages for using C. elegans as a model system to understand human aging. This review focuses on molecular genetics aspects of this model organism.     

用直接注射法生产转基因鱼

本文报道了对鲤鱼、鲫鱼受精卵不加任何去膜处理,用显微操作器把外源基因直接注射到卵核附近,构建转基因鱼的方法。本法操作方便,孵化条件简单,成活率高。斑点杂交和Southern Blot杂交结果表明,外源基因的整合率与其它方法构建的转基因鱼的外源基因的整合率相近。从1988年至今,本组运用这个方法生产转基因鲤鱼、鲫鱼一万余尾。     

Profiling the Anaerobic Response of C. elegans Using GC-MS

The nematode Caenorhabditis elegans is a model organism that has seen extensive use over the last four decades in multiple areas of investigation. In this study we explore the response of the nematode Caenorhabditis elegans to acute anoxia using gas-chromatography mass-spectrometry (GC-MS). We focus on the readily-accessible worm exometabolome to show that C. elegans are mixed acid fermenters that utilize several metabolic pathways in unconventional ways to remove reducing equivalents – including partial reversal of branched-chain amino acid catabolism and a potentially novel use of the glyoxylate pathway. In doing so, we provide detailed methods for the collection and analysis of excreted metabolites that, with minimal adjustment, should be applicable to many other species. We also describe a procedure for collecting highly volatile compounds from C. elegans. We are distributing our mass spectral library in an effort to facilitate wider use of metabolomics.     

Model-Independent Phenotyping of C. elegans Locomotion Using Scale-Invariant Feature Transform

To uncover the genetic basis of behavioral traits in the model organism C. elegans, a common strategy is to study locomotion defects in mutants. Despite efforts to introduce (semi-)automated phenotyping strategies, current methods overwhelmingly depend on worm-specific features that must be hand-crafted and as such are not generalizable for phenotyping motility in other animal models. Hence, there is an ongoing need for robust algorithms that can automatically analyze and classify motility phenotypes quantitatively. To this end, we have developed a fully-automated approach to characterize C. elegans’ phenotypes that does not require the definition of nematode-specific features. Rather, we make use of the popular computer vision Scale-Invariant Feature Transform (SIFT) from which we construct histograms of commonly-observed SIFT features to represent nematode motility. We first evaluated our method on a synthetic dataset simulating a range of nematode crawling gaits. Next, we evaluated our algorithm on two distinct datasets of crawling C. elegans with mutants affecting neuromuscular structure and function. Not only is our algorithm able to detect differences between strains, results capture similarities in locomotory phenotypes that lead to clustering that is consistent with expectations based on genetic relationships. Our proposed approach generalizes directly and should be applicable to other animal models. Such applicability holds promise for computational ethology as more groups collect high-resolution image data of animal behavior.     

谷氧还蛋白rbGrx可延缓CL4176秀丽隐杆线虫中β淀粉样蛋白诱导的毒性

阿尔茨海默病（Alzheimer’s disease,AD）是一种与衰老相关的神经退行性疾病,其中β-淀粉样蛋白（β-amyloid,Aβ）诱导的细胞毒性被认为是其发病的主要原因。本文以Aβ转基因秀丽隐杆线虫CL4176为模型,研究了重组荞麦谷氧还蛋白（recombinant buckwheat glutaredoxin, rbGrx）对Aβ诱导的毒性和氧化应激的影响。结果显示,4 μmol/L rbGrx可以延长CL4176线虫平均寿命达20%左右,并增加衰老虫体运动能力约43.6%,延迟产卵高峰期1 d,同时可以有效延缓Aβ毒性诱导的瘫痪表型。进一步研究发现,在正常条件和Aβ诱导毒性时,rbGrx均能降低CL4176线虫体内活性氧（reactive oxygen species,ROS）水平,并上调SOD活性和GSH含量。另外,rbGrx下调Aβ mRNA水平44.1％,减少Aβ沉积量,并且明显上调热激因子1 hsf-1（2.01倍）和hsp-16.2（2.65倍）mRNA表达水平。这表明,rbGrx通过降低CL4176线虫体内的ROS水平和上调热激蛋白质的转录表达水平,降低CL4176秀丽隐杆线虫中Aβ诱导的毒性。结果提示,rbGrx可能具有预防AD的潜力。     

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.     

秀丽隐杆线虫脂肪染色的方法比较(英文)

肥胖及其相关的疾病影响了越来越多的人.在哺乳动物中调节脂肪代谢的因子和脂肪代谢途径在线虫中也是保守存在的,因此线虫是脂肪研究的良好动物模型.在研究线虫脂肪代谢中,存在多种脂肪染色方法,而不同方法所表征的脂肪含量存在一定的差异,通过各种染色方法检测基因突变或者饥饿引起的脂肪含量变化.实验结果表明,采用油红、苏丹黑、尼罗红染料的固定染色法均能够正确地表征线虫的贮存脂肪.     

Generation of asymmetry and segregation of germ-line granules in early C. elegans embryos

Germ-line granules in C. elegans embryos (P granules) can be visualized by immunofluorescence microscopy using a monoclonal antibody. In mutant zygotes with abnormal spindle orientations and in wild-type zygotes treated with the microtubule inhibitors nocodazole, colcemid, vinblastine, and griseofulvin, both P-granule segregation to the posterior pole and the concomitant pseudocleavage occur apparently normally, but the normally concurrent migration of the pronuclei is inhibited. Conversely, treatment of wild-type embryos with the microfilament inhibitors cytochalasins D and B inhibits P-granule segregation and pseudocleavage, as well as other manifestations of polarity, without preventing pronuclear migration. The results suggest that P-granule segregation does not require either the spindle or cytoplasmic microtubules, but that this process as well as generation of other asymmetries does require cytoskeletal functions that depend on microfilaments.     

通过双原核显微注射提高转基因小鼠研制效率的实验研究

目的建立高效的转基因小鼠制备技术,为开展遗传工程动物模型研究奠定技术基础。方法通过向小鼠受精卵原核中注入不同浓度的DNA分子,筛选最适注射用DNA浓度;将K14/hCTLA4-Ig基因表达载体分子通过显微注射分别导入小鼠受精卵雌、雄原核,并设立单原核注射对照组;利用输卵管腹壶部穿刺移植法将注射后的小鼠受精卵移植于同期发情的受体母鼠;利用PCR对出生的转基因首建小鼠进行筛选。结果最适DNA分子浓度为10ng/μl;在单、双原核注射组胚胎2细胞卵裂率分别为52.3%(132/253)和45.0%(108/240),差异有显著性(P<0.05);注射胚胎移植后体内存活率分别为18.1%(24/132)和16.7%(18/108),差异无显著性;转基因首建小鼠阳性率分别为3/24和5/18,转基因阳性小鼠占总注射胚胎的比例为1.2%(3/253)和2.08%(5/240),差异有极显著性(P<0.01)。结论尽管双原核注射对胚胎的2细胞卵裂率有一定影响,但通过双原核注射可有效提高转基因小鼠的制备效率。     

显微注射法制备转基因小鼠的技术研究

转基因技术是发生工程学或胚胎操作技术中的一部分。这一技术包括有受精卵的采集,受精卵的体外培养,胚胎移植等技术,转基因动物制作是其应用技术之一,它是将外源ＤＮＡ片段用显微注射法直接注入受精卵原核,使外源ＤＮＡ随机整合到寄主基因组中而形成转基因小鼠。我们建立完善这一技术的目的是在实验动物的生产中为转基因动物的生产提供稳定可靠的保证。我们将ｐＣＸＴＧＡＰ、ｐＣＳＬＮ等基因分别注入受精卵原核制备相应的转基因动物模型,并从中总结较佳的操作技术。结果经检测阳性转基因小鼠占出生幼鼠的１６３８％。我们认为不仅要有精细的微注射技术、动物的选用和饲育、严密的胚胎操作都是转基因动物制作成功的重要条件。     

Comparing Platforms for C. elegans Mutant Identification Using High-Throughput Whole-Genome Sequencing

Background

Whole-genome sequencing represents a promising approach to pinpoint chemically induced mutations in genetic model organisms, thereby short-cutting time-consuming genetic mapping efforts.

Principal Findings

We compare here the ability of two leading high-throughput platforms for paired-end deep sequencing, SOLiD (ABI) and Genome Analyzer (Illumina; “Solexa”), to achieve the goal of mutant detection. As a test case we used a mutant C. elegans strain that harbors a mutation in the lsy-12 locus which we compare to the reference wild-type genome sequence. We analyzed the accuracy, sensitivity, and depth-coverage characteristics of the two platforms. Both platforms were able to identify the mutation that causes the phenotype of the mutant C. elegans strain, lsy-12. Based on a 4 MB genomic region in which individual variants were validated by Sanger sequencing, we observe tradeoffs between rates of false positives and false negatives when using both platforms under similar coverage and mapping criteria.

Significance

In conclusion, whole-genome sequencing conducted by either platform is a viable approach for the identification of single-nucleotide variations in the C. elegans genome.     

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.     

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode’s ATP levels.^1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.^4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.     

State-dependency in C. elegans

Memory and the expression of learned behaviors by an organism are often triggered by contextual cues that resemble those that were present when the initial learning occurred. In state-dependent learning, the cue eliciting a learned behavior is a neuroactive drug; behaviors initially learned during exposure to centrally acting compounds such as ethanol are subsequently recalled better if the drug stimulus is again present during testing. Although state-dependent learning is well documented in many vertebrate systems, the molecular mechanisms underlying state-dependent learning and other forms of contextual learning are not understood. Here we demonstrate and present a genetic analysis of state- dependent adaptation in Caenorhabditis elegans. C. elegans normally exhibits adaptation, or reduced behavioral response, to an olfactory stimulus after prior exposure to the stimulus. If the adaptation to the olfactory stimulus is acquired during ethanol administration, the adaptation is subsequently displayed only if the ethanol stimulus is again present. cat-1 and cat-2 mutant animals are defective in dopaminergic neuron signaling and are impaired in state dependency, indicating that dopamine functions in state-dependent adaptation in C. elegans.     

Innexins in C. elegans

Innexins are functionally analogous to the vertebrate connexins, and the innexin family of gap junction proteins has been identified in many invertebrates, including Drosophila and C. elegans. The genome sequencing project has identified 25 innexins in C. elegans. We are particularly interested in the roles that gap junctions may play in embryonic development and in wiring of the nervous system. To identify the particular C. elegans innexins that are involved in these processes, we are examining their expression patterns using specific antibodies and translational GFP fusions. In addition we are investigating mutant, RNAi and overexpression phenotypes for many of these genes. To date, we have generated specific antibodies to the non-conserved carboxyl termini of 5 innexins. We have constructed GFP translational fusions for 17 innexins and observed expression patterns for 13 of these genes. In total we have characterized expression patterns representing 14 innexins. Mutations have been identified in 5 of these genes, and at least 3 others have RNAi mutant phenotypes. Generalities emerging from our studies include: 1) most tissues and many individual cells express more than one innexin, 2) some innexins are expressed widely, while others are expressed in only a few cells, and 3) there is a potential for functional pairing of innexins.     

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.