lecture_Xenopus_notes

lecture_Xenopus_notes - Strategies to Perturb Development...

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1 Strategies to Perturb Development in Xenopus I. Introduction Important insight in gene function and developmental processes can be gained by perturbing normal development by specific manipulations. Xenopus is very accessible for such manipulations because of its external development, its size and the simple culture conditions. II. Administration of Reagents a. Administration via the Medium Some compounds can be administered via the medium (e.g. LiCl and Retinoic Acid). The prerequisite is that the compound can permeate the vitelline envelope and membrane of the embryo. This is essentially only through for small molecules. b. Microinjection Larger compounds, including RNA, DNA and proteins need to be microinjected (mostly by pressure-driven micro-injectors). To determine the injection volume, the glass capillary needle is either calibrated or a dye is supplemented to the injection liquid (e.g. 0.5% phenol red). Up to 20 nl can be injected into a blastomere of Xenopus. Interestingly, in Xenopus it is possible to distinguish at the 4-cell stage the axes (D-V, A-P) of the embryo and to predict the tissues to which a certain domain in the early embryo will contribute to later on (e.g. the dorsal animal site will contribute later to tissues like the brain and the eyes. Injections can be easily targeted to these specific regions in the embryo. c. Other Delivery Methods Electroporation has been described as an alternative method to deliver DNA into Xenopus zygotes but a more efficient method is sperm-mediated transfer. Spermatozoa are incubated with plasmid DNA (often in presence of a restriction enzyme) or electroporated prior to in vitro fertilization. This method is, however, not very efficient III. Reagents a. RNA RNA injections are widely used to achieve a widespread ectopic expression of a gene under study. Capped RNA is synthesized in vitro from a cDNA template. To increase the stability and translation efficiency of the injected RNA, the cDNA in the template is mostly flanked by 5’- and 3’ UTRs (untranslated regions) of the Xenopus β -globin gene and an SV40 polyadenylation site. Before cDNAs are
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2 cloned in the transcription vectors, their UTRs should be removed (since they may be destabilizing or highly regulated). Other issues increasing translation efficiency are Kozak sequences and the “cap”, a methylated guanylyl in a 5’-5’ linkage at the 5’ of the RNA. This can be linked to in vitro-syntesized RNA by adding a certain amount of cap analog, m 7 G(5’)ppp(5’)G to the transcription reaction. b. Oligonucleotides Oligos are mainly used for antisense approaches to target specific mRNAs. In zebrafish they can be applied via the medium (oligos can be modified). In Xenopus they need to be microinjected. Recently a special type of oligonucleotides have been generated, called
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lecture_Xenopus_notes - Strategies to Perturb Development...

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