Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre

被引:31
作者
Brecht, S [1 ]
Erdhart, H [1 ]
Soete, M [1 ]
Soldati, D [1 ]
机构
[1] Zentrum Mol Biol Heidelberg, D-69120 Heidelberg, Germany
关键词
apicomplexan parasite; excision; site-specific chromosomal integration; transformation;
D O I
10.1016/S0378-1119(99)00202-4
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38 kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34 bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids ape constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system. (C) 1999 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:239 / 247
页数:9
相关论文
共 27 条
[21]   A SITE-DIRECTED CHROMOSOMAL TRANSLOCATION INDUCED IN EMBRYONIC STEM-CELLS BY CRE-LOXP RECOMBINATION [J].
SMITH, AJH ;
DESOUSA, MA ;
KWABIADDO, B ;
HEPPELLPARTON, A ;
IMPEY, H ;
RABBITTS, P .
NATURE GENETICS, 1995, 9 (04) :376-385
[22]   EXPERIMENTAL INDUCTION OF BRADYZOITE-SPECIFIC ANTIGEN EXPRESSION AND CYST FORMATION BY THE RH STRAIN OF TOXOPLASMA-GONDII IN-VITRO [J].
SOETE, M ;
CAMUS, D ;
DUBRAMETZ, JF .
EXPERIMENTAL PARASITOLOGY, 1994, 78 (04) :361-370
[23]   TOXOPLASMA-GONDII - KINETICS OF BRADYZOITE-TACHYZOITE INTERCONVERSION INVITRO [J].
SOETE, M ;
FORTIER, B ;
CAMUS, D ;
DUBREMETZ, JF .
EXPERIMENTAL PARASITOLOGY, 1993, 76 (03) :259-264
[24]   A SELECTOR OF TRANSCRIPTION INITIATION IN THE PROTOZOAN PARASITE TOXOPLASMA-GONDII [J].
SOLDATI, D ;
BOOTHROYD, JC .
MOLECULAR AND CELLULAR BIOLOGY, 1995, 15 (01) :87-93
[25]   Processing of Toxoplasma ROP1 protein in nascent rhoptries [J].
Soldati, D ;
Lassen, A ;
Dubremetz, JF ;
Boothroyd, JC .
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1998, 96 (1-2) :37-48
[26]   Molecular genetic strategies in Toxoplasma gondii: Close in on a successful invader [J].
Soldati, D .
FEBS LETTERS, 1996, 389 (01) :80-83
[27]   Inducible site-directed recombination in mouse embryonic stem cells [J].
Zhang, Y ;
Riesterer, C ;
Ayrall, AM ;
Sablitzky, F ;
Littlewood, TD ;
Reth, M .
NUCLEIC ACIDS RESEARCH, 1996, 24 (04) :543-548