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Pubmed Article
High female survival promotes evolution of protogyny and sexual conflict.
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PLoS ONE
PUBLISHED: 03-17-2015
Existing models explaining the evolution of sexual dimorphism in the timing of emergence (SDT) in Lepidoptera assume equal mortality rates for males and females. The limiting assumption of equal mortality rates has the consequence that these models are only able to explain the evolution of emergence of males before females, i.e. protandry-the more common temporal sequence of emergence in Lepidoptera. The models fail, however, in providing adaptive explanations for the evolution of protogyny, where females emerge before males, but protogyny is not rare in insects. The assumption of equal mortality rates seems too restrictive for many insects, such as butterflies. To investigate the influence of unequal mortality rates on the evolution of SDT, we present a generalised version of a previously published model where we relax this assumption. We find that longer life-expectancy of females compared to males can indeed favour the evolution of protogyny as a fitness enhancing strategy. Moreover, the encounter rate between females and males and the sex-ratio are two important factors that also influence the evolution of optimal SDT. If considered independently for females and males the predicted strategies can be shown to be evolutionarily stable (ESS). Under the assumption of equal mortality rates the difference between the females' and males' ESS remains typically very small. However, female and male ESS may be quite dissimilar if mortality rates are different. This creates the potential for an 'evolutionary conflict' between females and males. Bagworm moths (Lepidoptera: Psychidae) provide an exemplary case where life-history attributes are such that protogyny should indeed be the optimal emergence strategy from the males' and females' perspectives: (i) Female longevity is considerably larger than that of males, (ii) encounter rates between females and males are presumably low, and (iii) females mate only once. Protogyny is indeed the general mating strategy found in the bagworm family.
Authors: Rudi L. Verspoor, Chloe Heys, Thomas A. R. Price.
Published: 04-22-2015
ABSTRACT
Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.
21 Related JoVE Articles!
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Assessing Differences in Sperm Competitive Ability in Drosophila
Authors: Shu-Dan Yeh, Carolus Chan, José M. Ranz.
Institutions: University of California, Irvine.
Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5. Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes. Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.
Developmental Biology, Issue 78, Molecular Biology, Cellular Biology, Genetics, Biochemistry, Spermatozoa, Drosophila melanogaster, Biological Evolution, Phenotype, genetics (animal and plant), animal biology, double-mating experiment, sperm competitive ability, male fertility, Drosophila, fruit fly, animal model
50547
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Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats
Authors: Lisa Y. Maeng, Kara K. Cover, Aaron J. Landau, Mohammed R. Milad, Kelimer Lebron-Milad.
Institutions: Massachusetts General Hospital, Harvard Medical School.
Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.
Behavior, Issue 96, estrogen, fear extinction, sex differences, estradiol, proestrus, metestrus, female, PTSD, anxiety
52202
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Flying Insect Detection and Classification with Inexpensive Sensors
Authors: Yanping Chen, Adena Why, Gustavo Batista, Agenor Mafra-Neto, Eamonn Keogh.
Institutions: University of California, Riverside, University of California, Riverside, University of São Paulo - USP, ISCA Technologies.
An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect’s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.
Bioengineering, Issue 92, flying insect detection, automatic insect classification, pseudo-acoustic optical sensors, Bayesian classification framework, flight sound, circadian rhythm
52111
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Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes
Authors: Atashi Sharma, Troy D. Anderson, Igor V. Sharakhov.
Institutions: Virginia Tech.
Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.
Infectious Diseases, Issue 95, Anopheles gambiae, malaria mosquito, DZNep, SAH, toxicological assay, epigenetics, vector control
52041
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Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice
Authors: Doris Tomas, Augustinus H. Prijanto, Emma L. Burrows, Anthony J. Hannan, Malcolm K. Horne, Tim D. Aumann.
Institutions: The University of Melbourne.
Long-lasting changes in the brain or ‘brain plasticity’ underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in ‘enriched environments’ (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of ‘DA’ genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson’s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).
Neuroscience, Issue 95, Behavior, midbrain, tyrosine hydroxylase, dopamine, plasticity, substantia nigra pars compacta
52329
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Production of Haploid Zebrafish Embryos by In Vitro Fertilization
Authors: Paul T. Kroeger Jr., Shahram Jevin Poureetezadi, Robert McKee, Jonathan Jou, Rachel Miceli, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.
Developmental Biology, Issue 89, zebrafish, haploid, in vitro fertilization, forward genetic screen, saturation, recessive mutation, mutagenesis
51708
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Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs
Authors: Sander van Iersel, Elferra M. Swart, Yumi Nakadera, Nico M. van Straalen, Joris M. Koene.
Institutions: VU University.
In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.
Physiology, Issue 88, Allohormone, Fresh-water snail, Gastropod, Lymnaea stagnalis, Mollusc, Pond snail, Prostate, Semen, Seminal fluid Sexual selection, Sperm
51698
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Barnes Maze Testing Strategies with Small and Large Rodent Models
Authors: Cheryl S. Rosenfeld, Sherry A. Ferguson.
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g. bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g. distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e. random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g. shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
51194
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Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis
Authors: Anastasia R. Nast, Cassandra G. Extavour.
Institutions: Harvard University.
The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.
Developmental Biology, Issue 85, Amphipod, experimental embryology, micromere, germ line, ablation, developmental potential, vasa
51073
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Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models
Authors: Devon S. Svoboda, Alysen Clark, David S. Park, Ruth S. Slack.
Institutions: University of Ottawa.
Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell’s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.
Developmental Biology, Issue 95, In utero electroporation, brain development, neural migration, transgenic, cell autonomous, mouse model
51983
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Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Authors: Stacy L. Fairbanks, Rebekah Vest, Saurabh Verma, Richard J. Traystman, Paco S. Herson.
Institutions: University of Colorado School of Medicine, Oregon Health & Science University, University of Colorado School of Medicine.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
Neuroscience, Issue 82, male, female, sex, neuronal culture, ischemia, cell death, neuroprotection
50758
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Measurement of Lifespan in Drosophila melanogaster
Authors: Nancy J. Linford, Ceyda Bilgir, Jennifer Ro, Scott D. Pletcher.
Institutions: University of Michigan , University of Michigan .
Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals. The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (http://sitemaker.umich.edu/pletcherlab/software). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.
Developmental Biology, Issue 71, Cellular Biology, Molecular Biology, Anatomy, Physiology, Entomology, longevity, lifespan, aging, Drosophila melanogaster, fruit fly, Drosophila, mortality, animal model
50068
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Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Authors: Martin De Vos, Georg Jander.
Institutions: Cornell University.
Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
Plant Biology, Issue 15, Annual Review, Plant Resistance, Herbivory, Arabidopsis thaliana, Pieris rapae, Caterpillars, Butterflies, Jasmonic Acid, Glucosinolates
683
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Molecular Evolution of the Tre Recombinase
Authors: Frank Buchholz.
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.
Cell Biology, Issue 15, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
791
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Making Gynogenetic Diploid Zebrafish by Early Pressure
Authors: Charline Walker, Greg S. Walsh, Cecilia Moens.
Institutions: University of Oregon, Fred Hutchinson Cancer Research Center - FHCRC.
Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.
Developmental Biology, Issue 28, Zebrafish, Early Pressure, Homozygous Diploid, Haploid, Gynogenesis
1396
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Microinjection of Medaka Embryos for use as a Model Genetic Organism
Authors: Sean R. Porazinski, Huijia Wang, Makoto Furutani-Seiki.
Institutions: University of Bath.
In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.
Developmental Biology, Issue 46, medaka , zebrafish, evolution, mutant, vertebrate, genome function
1937
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Automated Interactive Video Playback for Studies of Animal Communication
Authors: Trisha Butkowski, Wei Yan, Aaron M. Gray, Rongfeng Cui, Machteld N. Verzijden, Gil G. Rosenthal.
Institutions: Texas A&M University (TAMU), Texas A&M University (TAMU).
Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.
Neuroscience, Issue 48, Computer animation, visual communication, mate choice, Xiphophorus birchmanni, tracking
2374
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Assessment of Motor Balance and Coordination in Mice using the Balance Beam
Authors: Tinh N. Luong, Holly J. Carlisle, Amber Southwell, Paul H. Patterson.
Institutions: California Institute of Technology.
Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.
Neuroscience, Issue 49, motor skills, coordination, balance beam test, mouse behavior
2376
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Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury
Authors: Angelo C. Lepore.
Institutions: Thomas Jefferson University Medical College.
Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2. ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease. Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8. Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9. Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.
Medicine, Issue 55, cell transplantation, engraftment, graft, spinal cord, stem cells, precursors, ALS, amyotrophic lateral sclerosis, motor neuron, SCI, spinal cord injury
3069
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Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil
Authors: Danilo O. Carvalho, Derric Nimmo, Neil Naish, Andrew R. McKemey, Pam Gray, André B. B. Wilke, Mauro T. Marrelli, Jair F. Virginio, Luke Alphey, Margareth L. Capurro.
Institutions: Oxitec Ltd, Universidade de São Paulo, Universidade de São Paulo, Moscamed Brasil, University of Oxford, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM).
New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world. Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults11,12. To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.
Basic Protocol, Issue 83, Aedes aegypti, mass rearing, population suppression, transgenic, insect, mosquito, dengue
3579
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Harvesting Sperm and Artificial Insemination of Mice
Authors: Amanda R. Duselis, Paul B. Vrana.
Institutions: University of California, Irvine (UCI).
Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female’s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated. Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.
Developmental Biology, Issue 3, sperm, mouse, artificial insemination, dissection
184
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