Trypanosoma brucei: a first-generation CRE-loxP site-specific recombination system.
The bacteriophage CRE-loxP system of DNA recombination is widely used to manipulate segments of the genomes of mice and other eukaryotes for the purpose of studying the regulation and functions of their genes. Since this recombination system could have similar applications in analyzing the genomes of trypanosomatids, we assessed the action of CRE recombinase on its loxP DNA recognition sites in Trypanosoma brucei after inserting tetracycline-regulated CRE and two 34-bp loxP sites into the T. brucei genome. We found that when loxP sites flank in a direct orientation the transcription termination sequence (1.1 kb) of the T. brucei GPEET/PAG3 locus, CRE recombinase deletes this termination sequence, permitting transcription and subsequent expression of a downstream reporter gene for the green fluorescent protein (GFP). Thus, the CRE-loxP system is highly efficient in T. brucei, but the experimental results also indicate that a better way than the existing tetracycline-regulated system is required to completely silence expression of CRE in the T. brucei genome when it is not needed before the full range of CRE-loxP applications currently used in mice can be exploited in African trypanosomes.
Barrett
Brian
B
Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
LaCount
Douglas J
DJ
Donelson
John E
JE
D000818:Animals; D001483:Base Sequence; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics / Q000502:physiology; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D008164:Luminescent Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases