Transient and stable transfection of Leishmania by particle bombardment

Abstract

Transfection of trypanosomatids, particularly the transfection of their mitochondrion and different developmental stages, is still a limiting step in many experiments using recombinant DNA technology. Two types of trypanosomatid transfection protocols have been described so far. The standard protocol is based on electroporation first described by Bellofatto and Cross [1]. The second protocol is based on transfection using a homemade particle gun [2]. The biolistic method was first applied to plants [3], but is now routinely used for transfection of a wide variety of targets ranging from bacterial to mammalian cells [4–10] and it is the only method described so far to transfect organelles [10,11]. In trypanosomatids, the genetic organization of the mitochondrion shows several unusual features like a complex DNA network composed of thousands of catenated circular DNA molecules [12], the lack of encoded tRNAs [13] and the still enigmatic process of RNA editing [14]. However, the trypanosomatid mitochondrion is not yet accessible to recombinant DNA technology, and therefore the experimental in vivo analysis of the mentioned features is severely restricted. It is intended that the particle bombardment technique should be applied to transfect mitochondria of trypanosomatids. Since the optimal bombarding conditions for nuclear and organellar transfection of a given organism generally are identical [6,10,15,16] and as a first step, the factors influencing the nuclear transfection efficiency have been investigated. The ability of microprojectiles to deliver DNA into Leishmania cells was demonstrated either by the presence of transient b-glucuronidase (GUS) activity or by selection of stable transformants on plates following bombardment with the plasmid pX63NEO-GUS [17] using the DuPont Biolistic Abbre6iations: GUS, b-glucuronidase. * Corresponding author. Tel.: +41 31 6314342; fax: +41 31 6313383; e-mail beat.blum@ibc.unibe.ch