CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Abstract

The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei

“…Transgenic lines were selected by blasticidin and puromycin resistance, and correct insertion of the knock-out cassettes was assessed by PCR and Western blotting for expression of the Ty1 tag. The resulting conditional null cell lines were finally transfected with undigested pLEW100Cre_del_tetO (Addgene plasmid 24019; George Cross) for the transient expression of Cre recombinase and excision of the loxP-flanked knock-out cassettes, allowing reuse of drug resistance selectable markers (50,51). Cell lines that had lost the herpes simplex virus thymidine kinase gene were selected by resistance to ganciclovir (Invivogen).…”

Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei

“…PFs of wildtype Lister 427 were grown in SDM-79 (Brun and Schonenberger 1979) containing 10% fetal bovine serum and 0.25% hemin. Transfections were performed using a Nucleofector (Amaxa) as described previously (Scahill et al 2008).…”

Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: Proof of concept

“…The establishment of molecular tools that allow the inducible excision of large DNA segments to generate knockouts or to remove selectable markers is a remarkable necessity for improving molecular biology research on T. cruzi. The CRE recombinase system is a versatile tool in which the recombinase catalyzes the recombination between two loxP sites, thus promoting the excision or inversion of the loxP flanked DNA [4][5][6]. Despite its feasibility in several organisms, the CRE recombinase expression shows some degree of toxicity and it reinforces the necessity of a tight control of its expression.…”

“…In order to improve the CRE expression regulation, several modifications were introduced by Scahill and cols. [4]. Although there were some concerns about the regulated expression, the CRE recombinase system has been successfully used to manipulate gene expression or to remove selectable markers in T. brucei [4,6,7].…”

“…[4]. Although there were some concerns about the regulated expression, the CRE recombinase system has been successfully used to manipulate gene expression or to remove selectable markers in T. brucei [4,6,7]. These scenarios prompt us to test the regulatable CRE recombinase to achieve genomic manipulation in T.cruzi.…”