Product List for "Generation of Transgenic Xenopus laevis" Protocol

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View the protocol, "Generation of Transgenic Xenopus laevis" at BioSupplynet.com

Shoko Ishibashi1, Kristin L. Kroll2, and Enrique Amaya1,3

1 The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom 2 Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA 3 Corresponding author (enrique.amaya@manchester.ac.uk)

Generation of Transgenic Xenopus laevis: I. High-Speed Preparation of Egg Extracts. Please cite as CSH Protocols; 2007; doi:10.1101/pdb.prot4838.

ABSTRACT
Manipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes the method for the high-speed preparation of egg extracts. Briefly, a crude, cytostatic factor (CSF)-arrested egg extract (i.e., cytoplasm arrested in meiotic metaphase) is prepared. These extracts are driven into the interphase stage of the cell cycle by addition of calcium, and high-speed centrifugation is performed to obtain a purer cytoplasmic fraction. This fraction promotes swelling of sperm nuclei, but does not promote DNA replication. By adding the egg extract to the reaction, the sperm chromatin partially decondenses, facilitating integration of plasmid DNA into the genome.

Related Information:
For details on basic Xenopus procedures used in this protocol, please see Handling Xenopus laevis Adults, Inducing Ovulation in Xenopus laevis, Xenopus laevis Egg Collection, and Dejellying Xenopus laevis Embryos.

Products Available for this Protocol
Protocol Material Description	Product #	Product Name	Add to Cart
10% bovine serum albumin	A7906	Albumin from bovine serum, pH ~7 (1% in 0.15 M NaCl), ≥98% (agarose gel electrophoresis), lyophilized powder
Hoechst No. 33342	B2261	bisBenzimide H 33342 trihydrochloride, ≥98% (HPLC and TLC)
NaOH	655104	Sodium hydroxide, reagent grade, 97%, powder
Human chorionic gonadotropin (HCG)	C0434	Chorionic gonadotropin human, from human pregnancy urine, lyophilized powder
L-α-lysolecithin, egg yolk	L4129	L-α-Lysophosphatidylcholine from egg yolk, ~99%, Type I, powder
CaCl2	C2661	Calcium chloride, plant cell culture tested, anhydrous
KCl	P9541	Potassium chloride, for molecular biology, ≥99%
MgCl2	M4880	Magnesium chloride, insect cell culture tested
NaCl	S3014	Sodium chloride, for molecular biology, ≥98% (titration)
Dithiothreitol	D5545	DL-Dithiothreitol, SigmaUltra, >99% (titration)
EDTA (pH 8.0)	E7889	Ethylenediaminetetraacetic acid disodium salt solution, for molecular biology, 0.5 M
HEPES	H4034	HEPES, Biotechnology Performance Certified, ≥99.5% (titration), cell culture tested
Spermidine trihydrochloride	S2501	Spermidine trihydrochloride, ≥98% (TLC)
Spermine tetrahydrochloride	S1141	Spermine tetrahydrochloride, for molecular biology
Sucrose	S7903	Sucrose, SigmaUltra, pH 5.5–7.5 (20 °C, 1 M in H2O), >99.5% (GC)
Pregnant mare serum gonadotropin (PMSG)	G4877	Gonadotropin from pregnant mare serum, ≥1,000 IU/mg
Glycerol	G5516	Glycerol, for molecular biology, ≥99%
Tricaine methanesulfonate	E10521	Ethyl 3-aminobenzoate methanesulfonate, 98%
Sodium bicarbonate	S7277	Sodium bicarbonate, for molecular biology, 99.7–100.3%
Chymostatin	C7268	Chymostatin, microbial
DMSO	D2438	Dimethyl sulfoxide, Biotechnology Performance Certified, sterile-filtered, hybridoma tested, meets EP, USP testing specifications
Leupeptin	L9783	Leupeptin hydrochloride, microbial, ≥90% (HPLC)
Pepstatin	P5318	Pepstatin A, microbial, ≥90% (HPLC)
L-cysteine hydrochloride 1-hydrate	C121800	(R)-(+)-Cysteine hydrochloride hydrate
EGTA	E3889	Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, for molecular biology, ≥97%
ATP	A7699	Adenosine 5'-triphosphate disodium salt, SigmaUltra, ≥99%
Creatine phosphate	27920	Sodium creatine phosphate dibasic tetrahydrate, BioChemika, ≥98.0% (NT)

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