Hierarchical molecular events driven by oocytespecific. This prevents unnecessary surgeries in the case that the rna preparation is not good. For example, one frog ovary corresponds to mouse ovaries and western blots can be. Primirnas are processed to premirnas in the cell nucleus by the droshadgcr8 microprocessor complex gregory et al.
For example, one frog ovary corresponds to mouse ovaries and western blots can be performed with protein samples from just one oocyte or 1 ul of extract see below. Oocytes are stored in the abdominal cavity of adult females in clumps called ovarian lobes that include oocytes, connective tissue, blood vessels and follicle cells. The oocyte has long been used as a tool for the analysis of membrane channels and transporters peng et al. Discard the oocytes if they have dried out too much i. Ovochymase, a 30kda xenopus laevis egg serine activesite. Pioneering the xenopus oocyte and egg extract system. Involvement of protein acyltransferase zdhhc3 in maintaining. Hierarchical molecular events driven by oocytespecific factors lead to rapid and extensive reprogramming. Ovochymase, a xenopus laevis egg extracellular protease. The gel is placed next to a nitrocellulose or pvdf polyvinylidene. Monitoring gene expression in a single xenopus oocyte using. Xenopus embryo undergoes a timeregulated synchronized cell division in the early stages of its development and therefore is a useful in vivo model system for studying cell division. The beads were pelleted, washed four times with icecold lysis buffer, mixed with minimum volume of 4 sdspage sample buffer and processed through standard electrophoresis and.
There have been at least five major types of studies. Xenopus oocyte recovery and injection protocol haswell lab please use this protocolother protocols are for reference only experimental design surgery is not performed until the rna has been made and quantified. Two distinct aquaporin 0s required for development and. T1 a rapid western blotting protocol for the xenopus oocyte. Oocytes of the african clawed frog, xenopus laevis, are often used for expression and biochemical. Preparation of cell lysate from mouse oocytes for western.
Indeed, xenopus was the first vertebrate animal for which methods were developed to allow rapid analysis of gene function using misexpression by mrna injection. Western blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. Aven is dynamically regulated during xenopus oocyte. The cytosolic bmor1 was not detected, possibly due to quick degradation. A rapid western blotting protocol for the xenopus oocyte ncbi. Microinjection of xenopus laevis oocytes followed by thinsectioning electron microscopy em is an excellent system for studying nucleocytoplasmic transport. Here, we detail a rapid and convenient western blotting method for individual xenopus oocytes. Right panel a silver stained sdspage gel as a control for the western blot. Monitoring gene expression in a single xenopus oocyte using multiple cytoplasmic collections and quantitative rtpcr alexander a. Xenopus oocytes oxford academic journals oxford university.
Take the needle out of the oocyte and move the petri dish along to the next oocyte. Xenopus oocytes are arrested in prophase of meiosis i due to high protein kinase a pka activity. Xenopus embryos for in vivo studies of human disease gene function. Induction of maturationpromoting factor during xenopus. The roles of maternal vangl2 and apkc in xenopus oocyte and. Xenopus laevis isoform of g proteincoupled receptor 3. Drosha cleavage of microinjected primirnas is enhanced by maturation of xenopus laevis oocytes to eggs. Briefly, oocytes were lysed with a pipette in lysis buffer20 mm. The animal protocols used in this study adhered to the arvo statement for the. Microinjection of xenopus oocyte gene delivery group, university of birmingham this protocol covers. B western blot of xenopus egg extracts probed with the four different polyclonal affinitypurified antixklp2 antibodies used at 0. The initial step of thermotreatment rendered most of the extract a b c figure 2 activation of t7 rna polymerase t7 rnap in cellfree extracts of xenopus oocyte and eggs.
Briefly, oocytes were lysed with a pipette in lysis buffer20 mm tris, ph 7. The beads were pelleted, washed four times with icecold lysis buffer, mixed with minimum volume of 4 sdspage sample buffer and processed through standard electrophoresis and western blot protocol using icecold caps buffer 0. Electrophoresis and western blot each oocyte was lysed as a single cell in 10 l of the following homogenization bu er 50 mm hepes ph 7. Translational repression by the oocytespecific protein. Scientific protocols microinjection of xenopus oocyte. Western blotting is used to visualize proteins that have been separated by gel electrophoresis. Because of its large nucleus and high density of nuclear pore complexes npcs, nuclear transport can be easily visualized in the xenopus oocyte. Approximately 5 g of this extract was analyzed by a colorimetric western blot method using alkaline phosphatase. Xenopus oocyte microinjection northwestern university. Experimental setup for western blot analysis and transcriptional inhibition. Xklp2, a novel xenopus centrosomal kinesinlike protein. Downregulation of rna helicase iigu results in the. Furthermore, unlike most amphibians, oocytes and eggs could be obtained yearround from laboratorymaintained animals.
Twoelectrode voltageclamp recording from xenopus oocytes. Adult xenopus frogs can be purchased from nasco, inc. Heterologous protein expression in the xenopus oocyte. The method exploits recently introduced rapid blotting systems, commercially available from invitrogen iblot or biorad trans blot turbo.
For small scale preparations, germinal vesicles were manually iso. B bmor2 expression in the membrane fraction of xenopus oocytes. A kinetics of t7 rnap activation in oocyte and egg extracts. Xenopus was particularly attractive because the oocyte, egg, and embryonic cells were large enough that biochemically significant amounts of material could be microinjected into them. Limitations of using xenopus oocytes for expression of exogenous proteins.
In considering where to undertake postdoctoral training, i believed it was important to continue pioneering the xenopus oocyte and egg system. The oocyte should be smooth and moist if they have started to dry then, inject some of the sample over the injection site before attempting to insert the needle. T7 rnap was preincubated in the extracts at rt for the indicated. Western blot analysis also showed that the vast majority of the proteases in ovary, eggs, and embryos were present as the proenzyme forms, suggesting that the functions of these proteases depend on very limited levels of activation. Drosha protein levels are translationally regulated during xenopus oocyte maturation. Ovochymase, a xenopus laevis egg extracellular protease, is. The method exploits recently introduced rapid blotting systems, commercially available from. Fertilization of xenopus oocytes using the host transfer. The xenopus laevis oocyte offers an alternative system for. Western blot protocols magicmark xp western protein standard novex chromogenic substrates novex trisglycine midi gels quick reference western blotting using iblot 7minute blotting system western blotting using nitrocellulose membranes. Western blot analysislarge scale preparation of oocyte extract was carried out by freon extraction to remove yolk protein as described 37. Drosha protein levels are translationally regulated during. The material that xenopus can offer in biology is great in terms of quality and quantity. Antibodies and western blot analysis western blot analysis was performed as described previously 15.
The xenopus oocyte contains components of both the planar cell polarity and apicalbasal polarity pathways, but their roles are not known. Xenopus laevis oocytes, two ybox proteins, frgy2 and its homolog. Xenopus oocyte recovery and injection protocol haswell lab please use this protocolother protocols are for reference only experimental design. D western blot analysis of xenopus hira expression 48 h following injection of hira mrna to the oocytes. Xenopus embryos are large and easily manipulated, and moreover, thousands of embryos can be obtained in a single day. Electrical recordings of the mitochondrial calcium uniporter. Monitoring gene expression in a single xenopus oocyte. A,b schematic representation of a xenopus oocyte a and its microscopic image with the inserted sampling capillary during collection of cytoplasmic material b. Nuclear microinjection to assess how heterologously expressed. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Middle panel bmor1 expression in the cytosolic fraction. Protocol submitted by vgp from conlon lab protocols.
Plus indicates that the antigen was added in a 50100 times molar excess, and minus indicates that it was not. Effect of water hardness on oocyte quality and embryo. E qrtpcr analysis of transcription 24 h following the transfer. Godfrey, phd1, and george e sanders, dvm2,3 husbandry and health of the african clawed frog, xenopus laevis, greatly influences the quality of oocytes produced. A western blot protocol for detection of proteins heterologously expressed in xenopus laevis oocytes. Functional verification of membrane proteins in the oocyte. Microinjection of xenopus oocytes combined with thinsectioning em is a highly effective tool for studying nucleocytoplasmic transport. Effect of water hardness on oocyte quality and embryo development in the african clawed frog xenopus laevis earl w. Gripping too tightly is a common among beginners and will result in the oocyte being pulled away with the follicle intact.
Western blot protocols thermo fisher scientific us. Ripa buffer 100 ml can store at 4 add 790mg tris base to 75ml dih20. The roles of maternal vangl2 and apkc in xenopus oocyte. Preparation of the oocytes, selection of oocytes for microinjection. From gene to p r o t e i n by hermona soreq and shlomo seidman i. Growtharrestspecific protein 2 inhibits cell division in. Western blot analysis also showed that the vast majority of the proteases in ovary, eggs, and embryos were present as the proenzyme forms. Here, we examine the distribution, interactions and functions of the maternal planar cell polarity core protein vangl2 and the apicalbasal complex component apkc. The large oocyte nucleus germinal vesicle, gv is located in the animal hemisphere. We show that vangl2 is distributed in animally enriched islands in the subcortical cytoplasm. Activation of t7 rna polymerase in xenopus oocytes and cell. A western blot protocol for detection of proteins heterologously.
Pdf a rapid western blotting protocol for the xenopus oocyte. The protocol that follows includes the surgical procedure for extraction of. The lids should contain about 20 holes approximately 1 cm in diameter. Western blot analysis on membraneenriched fractions from xenopus oocytes. S1 western blot analysis of or expression in xenopus oocytes injected with the indicated crnas. Request pdf a western blot protocol for detection of proteins heterologously expressed in xenopus laevis oocytes oocytes of the african clawed frog, xenopus laevis, are often used for. Since immunoprecipitation and western blotting are standard techniques, in this section. A rapid western blotting protocol for the xenopus oocyte cold spring harb protoc. The initial step of thermotreatment rendered most of the extract a b c figure 2 activation of t7 rna polymerase t7 rnap in cellfree extracts of.
Electrical recordings of the mitochondrial calcium. This system has been used to map distinct steps of. A rapid western blotting protocol for the xenopus oocyte article pdf available in cold spring harbor protocols 203 march 20 with 587 reads how we measure reads. The level of bmor1 was higher in the membrane fraction when coexpressed with the or83b family. Sep 01, 2016 antibodies and western blot analysis western blot analysis was performed as described previously 15.
The tips should meet precisely and should be slightly blunter than forceps for making explants from xenopus embryos figure 1. S1 western blot analysis of or expression in xenopus oocytes. A rapid western blotting protocol for the xenopus oocyte. Activation of t7 rna polymerase in xenopus oocytes and. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Western blot analysis of oocyte lysates from all four conditions confirmed that flagtagged xgpr3 was appropriately expressed in oocytes injected with crna encoding the.
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