|
View the entire 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) |
|
Back to Top
View all available product lists for Cold Spring Harbor Laboratory Protocols
Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
|
