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(Chapter 10) Chromosome Engineering With Lambda-Integrase Mediated Recombination System - The ACE System

(Chapter 10) Chromosome Engineering With Lambda-Integrase Mediated Recombination System - The ACE System

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Published by: Conscience Science on Jul 26, 2013
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Chapter 10
Chromosome Engineering with Lambda-Integrase MediatedRecombination System: The ACE System
Tünde Praznovszky
Mammalian satellite DNA-based articial chromosomes (SATACs) are unique among the mammalianarticial chromosomes. These reproducibly generated de novo chromosomes are stably maintained indierent species, readily puried rom the host cell’s chromosomes and can be introduced into a variety o recipient cells. An articial chromosome expression system (ACE system) has been developed on theseSATACs to extend them or chromosome engineering. This system includes a Platorm ACE containingmultiple acceptor sites, specially designed targeting vector (ATV), and an ACE-integrase expression vec-tor (pCXLamIntROK). Gene o interest are cloned into targeting vector (ATV), and site-specic loadingo genes onto Platorm ACE is acilitated by ACE-integrase mediated recombination. ACE system is suit-able or multiple or subsequent loading o useul genes onto the same chromosome vector. This chapterdescribes the detailed procedure o chromosome engineering using the ACE system.
Key words:
Platorm ACE, Site-specic integration, Mutant lambda-integrase, Targeting vector ATV 
 Although genetic manipulation o mammalian cells and animalshave been achieved successully by plasmid and viral gene deliv-ery, mammalian articial chromosomes (MACs), as potential vec-tors oer signicant improvement in gene transer. MACs providestable, nonintegrating introduction o large payloads o geneticinormation, and or this reason several groups generated articialchromosomes using dierent approaches (1–12). We developed a satellite DNA-based articial chromosome(SATAC) (13–16). This chromosome had some basic advantagescompared to previously published MACs. The generation o SATACs was reproducible in a variety o host cells, they were
1. Introduction
Gyula Hadlaczky (ed.),
Mammalian Chromosome Engineering: Methods and Protocols
, Methods in Molecular Biology, vol. 738,DOI 10.1007/978-1-61779-099-7_10, © Springer Science+Business Media, LLC 2011
composed o known DNA sequences, and could be puried closeto 100% purity by high-speed fow cytometry. Several cell lines were established and also transgenic animals were produced usingisolated and puried SATACs (17).Disadvantage o SATAC-based gene delivery system was thatde novo generation o SATACs was necessary or each individualapplication. To overcome this problem, a mammalian articial chro-mosome engineering system (ACE system) was developed (18),based on lambda integrase catalyzed site/specic recombination.De novo SATAC was generated with multiple lambda inte-grase specic recognition/acceptor sites (Platorm ACE). A plas-mid construct pCXLamIntROK (pACE Integrase) providedintegrase expression or site-specic loading o exogenous DNA sequences into Platorm ACE. For targeting, a vector (ATV) wasconstructed, containing a lambda integrase recombination siteupstream o a promoterless selectable marker gene. In the courseo cotransection o Platorm ACE-carrying cell lines with ATV and pACE Integrase, site-specic integration o the ATV mole-cules took place (see Note 8). The selectable marker gene o ATV acquired a promoter located on the Platorm ACE, and cells car-rying correctly targeted ACE became resistant to the selectivedrug (Fig.1).Selected resistant clones were analysed by PCR using a primerpair specic to sequences o Platorm ACE and to the selectablemarker gene (Fig.2).Conventional, two-color fuorescent in situ hybridization(19) analyses were carried out exclusively on PCR screened, resis-tant clones. The integrity o ACE and site-specic integration onPlatorm ACE was also demonstrated (Fig.3). FISH on meta-phase spreads reveals not only the targeted integrations, butallows the detection o the nonspecic integration o the ATV inthe genome. In mouse cells, the eciency o targeting was morethan 90%, but in hamster cells it was usually below 50%.By the protocol provided here site-specic loading o useulgene(s) onto Platorm ACEs at 20–90% integration eciency wasachieved within a reasonably short period o time, i.e. in about 2months. In addition, the copy number o the introduced gene o interest can be variable on the loaded Platorm ACEs, allowingselection o cell lines with the desirable level o transgene expres-sion. Considering that Platorm ACE contains multiple acceptorsites, second round targeting o the already engineered ACE canbe achieved using the same protocol, with an ATV, carrying a di-erent promoterless selectable marker gene.This lambda integrase based chromosome engineering systemhas already been used successully to generate stable, high MAbexpressing CHO cell lines (20,21) and in a combined articial chromosome-stem cell gene therapy model experiment (22).
143Chromosome Engineering with Lambda-Integrase Mediated Recombination System
Fig. 1.
Site-specifc loading o a transgene onto the Platorm ACE 
. (
) The satellite DNA-based artifcial chromosome gener-ated by large-scale amplifcation contains multiple integration sites. (
) The recombination acceptor sites or the ACE-integrase, attP is located between a selectable marker gene and its promoter. (
) The ACE targeting vector (ATV) carries agene o interest bordered with insulator sequences, and the attB integrase specifc site upstream a promoterless select-able marker gene. (
) Expression o ACE-integrase catalyzes the recombination between attP and attB sites resulting in thesite-specifc integration o ATV into Platorm ACE. Thus, the promoterless selectable marker gene o ATV will be driven bythe promoter on Platorm ACE and drug resistance will be provided by the integrated marker gene o ATV.Fig. 2. Detection site-specifc integration in targeted cell lines by PCR. PCR was carried out using 193AF as the orwardprimer specifc to Platorm ACE and a reverse primer specifc to the selectable marker gene o ATV; the templates weregenomic DNAs o dierent transormed clones. Site-specifc targeting to Platorm ACE was detected in the genome oclones represented in lanes 1, 3, 4, and 5. In lane 2, the PCR showed no site-specifc integration o ATV. Lane 6 is thepositive, and lane 7 is the negative control o PCR, respectively. The Marker is the 1 kB DNA ladder (Fermentas).

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