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(Chapter 6) Rodent Transgenesis Mediated by a Novel Hyperactive Sleeping Beauty Transposon System

(Chapter 6) Rodent Transgenesis Mediated by a Novel Hyperactive Sleeping Beauty Transposon System

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87 
Gyula Hadlaczky (ed.),
Mammalian Chromosome Engineering: Methods and Protocols
, Methods in Molecular Biology, vol. 738,DOI 10.1007/978-1-61779-099-7_6, © Springer Science+Business Media, LLC 2011
Chapter 6
Rodent Transgenesis Mediated by a Novel HyperactiveSleeping Beauty Transposon System
Lajos Mátés
Abstract
DNA-based transposons are natural gene delivery vehicles. Similarly to retroviruses, these elementsintegrate into the chromosomes o host cells, but their lie-cycle does not involve reverse transcriptionand they are not inectious. Transposon-based gene delivery has several advantageous eatures comparedto viral methods; however, its ecacy has been the bottleneck o transposon utilization. Recently, usinga novel strategy or in vitro evolution, we created a new hyperactive version (SB100X) o the vertebrate-specic
Sleeping Beauty 
(SB) transposase. SB100X, when coupled with enhanced inverted terminal repeatstructure T2 type SB transposons, is over 100-old more active in mammalian cells than the prototype. We established protocol or SB100X mediated rodent transgenesis resulting on the average 35% trans-genic ounders with a low average number (1–2) o transgene insertions per ounder. Due to these char-acteristics the SB100X based protocol opens the possibility o designing SB based transgenes also orin vivo knockdown experiments. By the same token, single copy transgene units introduced by the SBtransposon system, more than being less prone to transgene silencing, also allow better control o trans-gene expression levels and patterns.
Key words:
Sleeping Beauty, SB100X, Transgenesis, Transgene silencing, In vivo knockdown
Class II transposons (also called DNA transposons) that move inthe host genome via a “cut-and-paste” mechanism are particu-larly useul tools or genome manipulations due to their easy lab-oratory handling and controllable nature. Schematic outlines o the structure and the transposition process o a class II transposonare presented in Fig.1a. Class II transposons are simply organized;they consist o a transposase-coding gene fanked by the invertedterminal repeats (ITRs). The ITRs contain the transposase bind-ing sites necessary or transposition. The process o transposition
1. Introduction
1.1. Class II (or DNA) Transposons 
 
88Mátés
can easily be controlled by separating the transposase source romthe transposable DNA harboring the ITRs, thereby creating anonautonomous transposon (Fig.1a). In such a two-componentsystem, the transposon can only be moved by supplementing thetransposase
in trans 
. In practice, any sequence o interest can bepositioned between the ITR elements according to the experi-mental needs. Transposition will result in excision o the elementrom the donor vector and subsequent integration into a new sequence environment (Fig.1b).Classic ways to induce expression o oreign genes in vertebratesrely on microinjection o nucleic acids into oocytes or ertilizedeggs. Two main limitations o these approaches are low rates o genomic integration and that the injected DNA generally inte-grates as a multicopy transgene concatemer. Both drawbacks canbe circumvented by utilizing transposition mediated gene deliv-ery because it can increase the eciency o chromosomal integra-tion and results in single-copy insertion events. Single-unitexpression cassettes are less prone to transgene silencing than are
1.2. Transposon- Mediated Transgenesis 
Fig. 1.
Cut and paste DNA transposition 
. (
a
) Scheme o a class II (or DNA) transposon,and that o a binary transposition system created by dissecting the transposasesource rom the transposon. (
b
) Outline o the mechanism o the “cut and paste”transposition.
 
89Rodent Transgenesis Mediated
the concatemeric insertions created using classical methods. Another particular problem concerning transgenesis is that ound-ers that develop rom the injected oocytes or eggs are predomi-nantly mosaic or the transgene because integration generally occurs relatively late during embryonic development. For exam-ple, in zebrash transgenesis, the mosaicism extent is high, due tothe integration o the transgene into the chromosome at latercleavage stages o the embryos (1, 2). Thereore, many transgenicounders transmit the transgene to only a ew percent o theirospring or do not transmit at all. In contrast, mosaicism doesnot seem to be a routine problem in rodents, where the sameintegration events predominantly happen at the one-cell stage ininjected embryos. Transposon-mediated transgenesis catalyzedby delivery o DNA encoding the transposase
in trans 
aces thesame transgene mosaicism problem because the oocytes are in atranscriptionally quiescent state and the embryonic genome acti- vation (EGA) starts species-specically at dierent stages ater theertilization. In mice, the major onset o transcription, EGA,begins during the 2-cell stage (3); it begins during the 4-cell stagein rats (4), and during the 8-cell to 16-cell stage in cattle (5). However, co-injection o engineered transposons with in vitrotranscribed transposase messenger RNA (mRNA) helps to over-come this limitation because only translation o the synthesizedmRNA is necessary to produce the transposase protein. By thisapproach it is possible to shit the window o transposon-mediatedintegration events to early stages in order to promote lower mosa-icism and successul transmission o the transgene to the nextgeneration in spite o the actual transcriptional quiescent state o the zygote. Currently the Sleeping Beauty (SB), piggyBac (PB),and Tol2 transposon systems are predominantly harnessed ortransgenic purposes in vertebrates due to their sucient activity in the vertebrate hosts. Lately, the mRNA co-injection method, where the in vitro synthesized mRNA o the transposase is co-injected with the transposon DNA, became the procedurepredominantly used or transposon-mediated transgenesis. Thismethod has been employed to generate transgenic zebrash withTol2 (6) and SB (7); transgenic Xenopus with SB (8) and Tol2 (9); and transgenic mice with SB (10) and more recently with a novel improved version o the SB transposase, SB100X (11).In case o animal transgenesis, a single copy transgene inser-tion not disturbing endogenous gene unctions is desirable. Theinsertional spectrum o the SB transposon system satises this cri-terion the best, because it integrates randomly at the genomelevel, and do not exhibit pronounced bias or integration intogenes or 5
¢
regulatory regions (12,13) . However, PB and Tol2 tolerate bigger cargo sizes (14, 15), which can be impor-tant or certain transgenes. Thereore the transposon systemshould be selected careully based on the actual experimental design.

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