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Genetic Manipulation of Kinetoplastida C.E

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Genetic Manipulation of Kinetoplastida C.E Reviews Genetic Manipulation of Kinetoplastida C.E. Clayton During the 1980s, many kinetoplastid genes were cloned promoter is required (Fig. 1b). There is as yet no repro- and their function inferred from homology with genes from ducible evidence for specific, strong initiation by RNA other organisms, location of the corresponding proteins or polymerase II in trypanosomatids (with the possible expression in heterologous systems. Up until 1990, before exception of the spliced leader loci), but RNA polym- the availability of DNA transfection methodology, we could erase I promoters [EP and variant surface glycoprotein not analyze the function of kinetoplastid genes within the (VSG) promoters in African trypanosomes, ribosomal organisms themselves. Since then, it has become possible to RNA promoters in all trypanosomatids] have proved create and complement mutants, to overexpress foreign pro- very useful5,6. The RNA polymerase I promoters are teins in the parasites, to knock out genes and even to switch species specific: hardly any activity is obtained if a vec- off essential functions. However, these methods are not tor containing a promoter from one trypanosomatid equally applicable in all parasites. Here, Christine Clayton species is transfected into another5,6 (C.E. Clayton et al., highlights the differences and similarities between the most unpublished). So far, nobody has obtained translatable commonly used model organisms, and assesses the relative mRNA from transcription driven by RNA polymerase advantages of different approaches and parasites for different III or spliced leader gene promoters. types of investigation. Permanent transformation using episomes The breakthrough in transfection technology started Transient assays are subject to interexperiment vari- with the transient expression of reporter genes after ability and involve high consumption of plasmid DNA electroporation of parasites with circular vectors1 and and cuvettes. Because only a small fraction of cells (often has since developed to include a wide spectrum of 2% or less) express the product of interest7, it is almost methods for functional gene analysis. impossible to study the localization of introduced gene products by electron microscopy, and difficult to ob- Transient transfections tain sufficient RNA product for quantitation or struc- Transient transfections are very useful for rapid tural analysis. Therefore, the creation of permanently analysis of a variety of functions such as transcription transformed cell lines has become the preferred route for initiation, post-transcriptional regulation and protein most applications. To achieve this, a selectable marker targeting. The minimal vector for transient gene ex- is added to the transient transfection vector (Fig. 1c). pression in Leishmania consists of a circular plasmid Commonly used markers and some possible alterna- containing a signal for trans-splicing of the mRNA, fol- tives are listed in Table 1. lowed by a reporter gene2. Better expression can often In all trypanosomatids, apart from the salivarian be obtained if the reporter gene is followed by a 39- species, stable transformants can be selected easily untranslated region (39-UTR) from a highly expressed after transformation of the cells with circular plasmids. gene. Because polyadenylation and trans-splicing are Usually, these multimerize to form bigger circles8; only coupled, this 39-UTR should be followed by a second Crithidia fasciculata retains monomeric plasmids of under trans-splicing signal (Fig. 1a). Promoters and RNA pro- 10 kb9. Expression of genes from episomes can be en- cessing signals from higher eukaryotes do not function hanced by increasing the concentration of selective drug, at all in trypanosomatids. Processing and expression resulting in amplification of the plasmid copy number are most efficient when 59-segments and 39-segments in the cells (see for example Refs 4,10). The mechanism from the homologous species are used (C.E. Clayton of transcriptional initiation in such episomes is ob- et al., unpublished). Developmental regulation of gene scure. For at least one example there was evidence for expression is mediated almost exclusively at the post- transcription of both DNA strands; strand preference transcriptional level; in most examples examined so far might be determined by terminators11. In T. cruzi, ex- the 39-untranslated region of the RNA determines the pression from episomes can be enhanced if a ribosomal RNA stability and translation efficiency. For example, RNA promoter is present12. In general, circular epi- the 39-untranslated regions of the genes encoding the somes are unstable in the absence of drug pressure, but major surface proteins of procyclic trypanosomes (EP1 stable recombinants are formed if the circles integrate and EP2, previously called PARP or procyclin) promote into the genome. high expression in procyclic-form Trypanosoma brucei and Transfection of T. brucei with circular plasmids very low expression in bloodstream forms3; that of the (with or without promoters) very seldom yields drug- A2 gene of Leishmania major gives amastigote-specific resistant parasites. In the rare exceptions, the DNA has expression4. either built an episome containing multiple tandem re- Although promoterless plasmids give readily detect- peats of the plasmid13,14 or the plasmid has recombined able expression in Leishmania, Crithidia and Trypanosoma into the genome. Two groups have selected for sequences cruzi, they are useless for transient assays in African tryp- capable of promoting maintenance of episomes in T. anosomes. In these parasites, a specific transcriptional brucei (reviewed in Ref. 15). Although stability-promoting sequences were found, there was no evidence that they Christine E. Clayton is at the Zentrum für Molekulare Biologie served as origins of replication, and the presence of (ZMBH), Im Neuenheimer Feld 282, Postfach 106249, D-69120 a EP promoter in the plasmids appeared to be obliga- Heidelberg, Germany. Tel: +49 6221 54 6876, Fax: +49 6221 tory. Episomal vectors and their uses are reviewed in 54 5894, e-mail: [email protected] Ref. 16. 372 0169-4758/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0169-4758(99)01498-2 Parasitology Today, vol. 15, no. 9, 1999 Reviews Permanent transformation by integration To promote integration of DNA into the genome, the input vector is linearized by restriction enzyme di- gestion within sequences identical to the proposed integration site17. In the examples shown (Fig. 1c,d) the region upstream of the first open reading frame is used for targeting. Non- homologous recombination of intro- duced DNA has not been reported. If several genomic targets are present, those of highest homology with the in- put plasmid are preferred18. In most cases it is advantageous to clone trans- fectants (on agarose or in microtiter plates) directly during the selection procedure, to ensure survival of trans- formants showing mildly deleteri- ous phenotypes that result in slowed growth. If a plasmid is targeted into a region of the DNA that is transcribed by RNA polymerase II, the whole integrated sequence is transcribed by read- Fig. 1. A selection of transfection vectors. Crucial elements are not to scale and are through from upstream. To obtain a color coded: promoter, red with red arrows indicating the direction of transcription; higher level of transcription that is 59-region, dark green; reporter gene or gene of interest, blue; 39-region, light green; se- reliably dependent on an added pol- lectable marker, purple; silent intergenic region, white. The plasmid backbone is shown ymerase I promoter, the plasmid must as a black line. Types of vector, with examples, are: simple promoterless transient trans- be integrated into a silent region of fection vector (a); transient vector with promoter (b); selectable promoterless ex- the genome, such as the ribosomal pression vector (c). The arrow indicates a site of cleavage that can be used to generate linear plasmid suitable for homologous genomic integration; selectable vector (shown RNA spacer14 or the spliced leader ar- 19 in c) integrated into the genome after cleavage at the site indicated by the arrows ray spacer (Fig. 1e). In T. brucei, tran- (d); promoter-bearing expression vector integrated into a silent portion of the genome scription of a reporter gene by RNA (e); plasmid with two promoters integrated into a silent portion of the genome (f). polymerase I results in 10–50 times higher expression than read-through transcription by RNA polymerase II14,19. If the expres- some promoter with the bacteriophage T7 promoter, sion obtained is not adequate, it might be useful to look and transfecting the construct into transgenic parasites first at the region 39 of the gene to be expressed; as that stably express bacteriophage T7 polymerase7,20. already mentioned, different 39-regions can give very Although the T3 promoter and polymerase can also be different levels of expression according to the life cycle used, the expression levels are lower than those seen stage. An additional order of magnitude increase in with T7, and we have found that they decline upon transcription can be obtained by replacing the trypano- prolonged passage7,19. Table 1. Markers available for selecting permanently transformed trypanosomes and Leishmaniaa Selectable marker Selection Drug target Mode of resistance Refs Neomycin phosphotransferase G418 Ribosome Enzymatic inactivation of drug 23, 46– 48 (NEO) Hygromycin
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