Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18% raffinose and 3% skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
Here we demonstrate a newly developed I•Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (> 60%) and rapid progressive motility (> 45%) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later recovered by IVF.
22 Related JoVE Articles!
Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos
Institutions: Universitat Autonoma de Barcelona.
Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1
and Wilmut 2
separately described that mouse embryos could be frozen and stored at -196 °C and, a few years later, Parkening et al
reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4
. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5
. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.
Developmental Biology, Issue 25, Cryopreservation, freezing, thawing, oocytes, embryos
Nuclear Transfer into Mouse Oocytes
Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).
Developmental Biology, Issue 1, oocytes, nuclear transfer, stem cells
Visualizing RNA Localization in Xenopus Oocytes
Institutions: Brown University.
RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis
oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo
, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout.
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.
Developmental Biology, Issue 35, RNA, Developmental Biology, Microinjection, RNA Localization, Xenopus, oocytes, VLE
Fertilization of Xenopus oocytes using the Host Transfer Method
Institutions: University of Iowa.
Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus
and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus
, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.
Developmental Biology, Issue 45, Xenopus, oocyte, host-transfer, fertilization, antisense
Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
Institutions: Instituto de Biotecnología-Universidad Nacional Autónoma de México, Edison State College.
Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+
-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+
dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.
Cellular Biology, Issue 75, Medicine, Molecular Biology, Genetics, Biophysics, Anatomy, Physiology, Spermatozoa, Ion Channels, Cell Physiological Processes, Calcium Signaling, Reproductive Physiological Processes, fluorometry, Flow cytometry, stopped flow fluorometry, single-cell imaging, human sperm, sperm physiology, intracellular Ca2+, Ca2+ signaling, Ca2+ imaging, fluorescent dyes, imaging
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
Institutions: University of Birmingham.
Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo
transplantation. In vivo
transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.
Cellular Biology, Issue 43, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
Production of Haploid Zebrafish Embryos by In Vitro Fertilization
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
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization
Institutions: University of California, Davis, Fred Hutchinson Cancer Research Center - FHCRC.
This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982). We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity. First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes. The rates of sperm freezing and thawing (δ°C/time) are probably the two most critical variables to control in this procedure. For this reason, do not substitute different tubes for those specified. Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs. Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.
Developmental Biology, Issue 29, Zebrafish, sperm, cryopreservation, TILLING
Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model
Institutions: United States Department of Agriculture, University of Maryland.
The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona
are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.
Basic Protocols, Issue 84, blastocyst, chimera, lentivirus, uterine transfer, oviductal transfer, utero-tubal transfer
Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments
Institutions: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU).
The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis
oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami
) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.
Biochemistry, Issue 89, two-electrode voltage-clamp, electrophysiology, biotinylation, Xenopus laevis oocytes, epithelial sodium channel, ENaC, proteases, proteolytic channel activation, ion channel, cleavage sites, cleavage fragments
Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro
Institutions: The University of Western Ontario, Children's Health Research Institute.
Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro
based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15
, and Scp3
. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.
Stem Cell Biology, Issue 77, Developmental Biology, Cellular Biology, Molecular Biology, Bioengineering, Biomedical Engineering, Medicine, Physiology, Adult Stem Cells, Pluripotent Stem Cells, Germ Cells, Oocytes, Reproductive Physiological Processes, Stem cell, skin, germ cell, oocyte, cell, differentiation, cell culture, mouse, animal model
A Method for Microinjection of Patiria minata Zygotes
Institutions: Carnegie Mellon University.
Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata
, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus
and Lytechinus variegatus
. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata
, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.
Developmental Biology, Issue 91, Embryology, Patiria miniata, sea star, echinoderm, development, gene regulatory networks, microinjection, gene expression perturbation, antisense oligonucleotide, reporter expression
Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification
Institutions: RIKEN BioResource Center.
Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1
, then followed by vitrification methods developed in the late 1980s2
. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3
, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5
. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.
Developmental Biology, Issue 57, mouse, embryo, cryopreservation, ethylene glycol, vitrification
Single Oocyte Bisulfite Mutagenesis
Institutions: Schulich School of Medicine and Dentistry, University of Western Ontario, Schulich School of Medicine and Dentistry, University of Western Ontario, Children's Health Research Institute.
Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1
. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2
. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3
. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6
towards ones that are more sensitive and reproducible7
. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8
. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9
. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10
. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11
, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12
. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.
Genetics, Issue 64, Developmental Biology, Biochemistry, Bisulfite mutagenesis, DNA methylation, individual oocyte, individual embryo, mouse model, PCR, epigenetics
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
Institutions: Technical University of Berlin, Oregon Health & Science University.
Whereas cation transport by the electrogenic membrane transporter Na+
-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+
-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+
transport by Na+
- or gastric H+
-ATPase in single cells. Using Xenopus
oocytes as expression system, we determine the amount of Rb+
) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+
uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g.
to quantitatively determine the 3Na+
transport stoichiometry of the Na+
-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+
-ATPase. In principle, the assay is not limited to K+
-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.
Biochemistry, Issue 72, Chemistry, Biophysics, Bioengineering, Physiology, Molecular Biology, electrochemical processes, physical chemistry, spectrophotometry (application), spectroscopic chemical analysis (application), life sciences, temperature effects (biological, animal and plant), Life Sciences (General), Na+,K+-ATPase, H+,K+-ATPase, Cation Uptake, P-type ATPases, Atomic Absorption Spectrophotometry (AAS), Two-Electrode Voltage-Clamp, Xenopus Oocytes, Rb+ Flux, Transversely Heated Graphite Atomizer (THGA) Furnace, electrophysiology, animal model
A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System
Institutions: Northwestern University, Northwestern University, Feinberg School of Medicine, Northwestern University, Northwestern University, Northwestern University.
The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro
follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4
. Our in vitro
culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5
. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8
. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9
. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10
. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5
. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro
culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11
. The degradable component may be particularly critical for clinical translation in order to support the greater than 106
-fold increase in volume that human follicles normally undergo in vivo
Bioengineering, Issue 49, Ovarian follicle, fibrin-alginate, 3D culture system, dynamic environment
Mouse Genome Engineering Using Designer Nucleases
Institutions: University of Zurich, University of Minnesota.
Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro
transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.
Genetics, Issue 86, Oocyte microinjection, Designer nucleases, ZFN, TALEN, Genome Engineering
Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes
Institutions: Stanford University , Stanford University School of Medicine.
Since its development by Sakmann and Neher 1, 2
, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.
Cellular Biology, Issue 20, Electrophysiology, Patch Clamp, Voltage Clamp, Oocytes, Biophysics, Gigaseal, Ion Channels
Live Imaging of GFP-labeled Proteins in Drosophila Oocytes
Institutions: Vassar College.
oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4
. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5
and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.
Developmental Biology, Issue 73, Biochemistry, Genetics, Cellular Biology, Molecular Biology, Proteins, Anatomy, Physiology, Drosophila melanogaster, fruit fly, Cell Biology, Drosophila oocytes, oogenesis, oocytes, ovaries, GFP, Live Imaging, Time Lapse Video, imaging, confocal microscopy, dissection, animal model
Microinjection of Xenopus Laevis Oocytes
Institutions: University of British Columbia - UBC.
Microinjection of Xenopus laevis
oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus
oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Panté, 2006). In addition, we have used microinjection of Xenopus
oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus.
Here we demonstrate the cytoplasmic microinjection of Xenopus
oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus
(AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.
Cellular biology, Issue 24, nuclear import, nuclear pore complex, Xenopus oocyte, microinjection, electron microscopy, nuclear membrane, nuclear import of viruses
Retrieval of Mouse Oocytes
Institutions: University of California, Irvine (UCI).
To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology. Together with the Peromyscus Genetic Stock Center (https://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system. A primary goal has been to optimize oocyte/early embryo retrieval.
Developmental Biology, Issue 3, oocyte, egg, mouse, dissection