Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.
19 Related JoVE Articles!
LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations
Institutions: The Hebrew University of Jerusalem, Ohio University.
Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1
, plants2, 3
, arthropods4, 5
, and bacteria7
. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10
, see Figure 1
. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11
, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10
, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2
. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3
. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4
. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis
. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10
. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13
. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15
. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13
. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16
The Cold Stage System
The cold stage assembly (Figure 2
) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 μm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).
Biochemistry, Issue 72, Chemistry, Physics, Biophysics, Bioengineering, Microbiology, Proteins, Ice binding proteins, IBP, antifreeze proteins, thermal hysteresis proteins, TH, ice structuring proteins, recrystallization inhibition proteins, nanoliter osmometer, LabVIEW, temperature control, microscopy stage, nano, Gibbs-Thomson effect, microfluidics, microscale chemistry
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
Institutions: Baylor College of Medicine, Baylor College of Medicine, University of California at San Diego, Baylor College of Medicine.
Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.
Neuroscience, Issue 84, Neurons, Cryo-electron Microscopy, Electron Microscope Tomography, Brain, rat, primary neuron culture, morphological assay
Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT
Institutions: Whitehead Institute for Biomedical Research, National University of Singapore, Massachusetts Institute of Technology.
Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1
. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR α- and β-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii
can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.
Developmental Biology, Issue 43, SCNT, immunology, TCR, BCR, mouse model, transnuclear
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
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
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry
Institutions: Washington University in St. Louis.
The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV
1.5), expressed in Xenopus
oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV
1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1
. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.
Developmental Biology, Issue 85, Voltage clamp, Cut-open, Oocyte, Voltage Clamp Fluorometry, Sodium Channels, Ionic Currents, Xenopus laevis
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
Mouse Oocyte Microinjection, Maturation and Ploidy Assessment
Institutions: University of Pennsylvania.
Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro
culture conditions 3
. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro
is a more economical and time-saving strategy.
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5
cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro
maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6
. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8
. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9
thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.
Cell biology, Issue 53, oocyte, microinjection, meiosis, meiotic maturation, aneuploidy
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
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
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
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
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
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
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
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 (http://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
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