Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

SHORT COMMUNICATION

Colony Polymerase Chain Reaction of Stably Transfected Trypanosoma cruzi Grown on Solid Medium

Vol. 95(1): 111-114, Jan./Feb. 2000

Wagner G dos Santos⁺⁺, Ivelina Metcheva, Gregory A Buck⁺

Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Box 980678 Richmond, VA 23298-0678, USA

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

Key words: Trypanosoma cruzi - stable transfection - Green Fluorescent Protein - neomycin phosphotransferase

Molecular studies of kinetoplastid parasites have revealed many unique mechanisms of gene regulation, gene expression and RNA processing in these organisms (Borst 1986, Vanhamme and Pays 1995). However, a general lack of efficient DNA transfection systems has precluded genetic dissection of these novel genetic processes. The development of transfection systems as a tool for genetic analysis of protozoan parasites has initiated a new phase in the study of these organisms, and both transient and stable transfection systems have been successfully employed to introduce and express foreign or modified genes. Among other applications, these transfection systems have been used: (i) to identify and characterize transcription promoters such as the rRNA and SL gene promoters in Trypanosoma cruzi (Tyler-Cross et al. 1995, Nunes et al. 1997a,b) and the PARP and VSG promoters in T. brucei (Zomerdijk et al. 1990, Rudenko et al. 1990), (ii) to functionally complement selected endogenous genes (Kelly et al. 1994, Nozaki & Cross 1994) and, (iii) to analyze gene function based on inactivation by targeted gene disruption (Lee & Van der Ploeg 1990, Otsu et al. 1993, Cooper et al. 1993, Chung et al. 1994).

In many cases after transfection of the parasite, it is necessary to select and clone the transformants before further analysis. For T. cruzi, this cloning has been done primarily by the extremely time consuming and uncertain process of limiting dilution and subculture in liquid medium. Only recently, an optimized method to isolate transformants from solid medium was reported (Mondragon et al. 1999). Herein, we report a simple method to select stable transfected T. cruzi epimastigotes grown on solid medium and to analyze the genotype by using colony polymerase chain reaction (PCR).

We have constructed a plasmid, pPAGFPAN, which contains the T. cruzi rRNA promoter cloned upstream from the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria (Kain et al. 1995) and the bacterial neomycin phosphotrans-ferase gene (NEO) (Fig. 1). To be properly processed by trans-splicing the splice acceptor sites 35.1 SAS and 35.2 SAS (McCarthy-Burke et al. 1989) were inserted upstream of NEO and GFP genes, respectively. Transfection of T. cruzi epimastigotes strain CL-brener with this plasmid generated a neomycin resistant (neo^r) population, which contained parasites co-expressing GFP (Fig. 2). After plating these parasites in blood-agar-LIT solid medium supplemented with 300 µg/ml of G418 and incubating the plate at 28°C for 20-30 days, single isolated ~1 mm² opaque colonies appeared (Fig. 3). Although the plating efficiency of these transfected parasites was quite low (~0.005), these colonies represent clones of the stably transfected T. cruzi.

To verify the presence of the reporter gene (GFP) in these T. cruzi colonies, colony PCR was performed. Several colonies were picked using a sterile plastic pipette tip, and transferred to 20 µl of sterile double distilled H₂O. These T. cruzi suspensions were incubated at 100°C for 5 min, vortexed vigorously for 2 min to release the nucleic acids, and the cellular debris was removed by centrifugation for 2 min at top speed in a microcen-trifuge. The supernatant was removed and transferred to a clean microcentrifuge tube and 10 µl was used as DNA template in PCR reactions with GFP and NEO-specific primers (Fig. 3) flanking either the NEO or GFP gene. All colonies examined showed a 700 bp PCR fragment derived from the GFP gene and a 800 bp fragment derived from the NEO gene (Fig. 3).

At the same time the colonies were picked for PCR analysis, an aliquot was also picked to inoculate 5 ml of LIT medium (Camargo 1964) supplemented with inhibitory concentrations (500 µg/ml) of G418. These cultures were incubated for seven days, and 1 ml was pelleted by centrifugation in a microcentrifuge tube. The pellet of T. cruzi epimastigotes was resuspended in 20 µl of sterile water and subjected to PCR analysis as described above. PCR product with size consistent with that expected for NEO and GFP genes were observed, confirming the presence of the plasmid in the cultured parasites (Fig. 3).

This work provides an important tool for the genetic manipulation of T. cruzi and for monitoring the genotype of transfected T. cruzi. Thus, transfected epimastigotes were selected and cultured as clonal colonies on solid medium supplemented by inhibitory concentrations of G418. This approach for cloning transformed T. cruzi represents significant advantages of time and effort over the limited dilution approach previously available, and will facilitate more the ongoing genetic dissection of these important pathogens.

REFERENCES

Fig. 1 | Fig. 2 | Fig. 3

This work was supported by grants from the National Institutes of Health, the American Heart Association, and The Jeffress Memorial Trust. WGS was supported by a fellowship from CNPq.

⁺Corresponding author. Fax: [phone]. E-mail: [e-mail].

⁺⁺Current address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande nº 6, Apartado 127, Oeiras, 2780-Portugal.

Received 18 August 1999

Accepted 11 November 1999

Figure 1

Figure 2

Figure 3

• Borst P 1986. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem 55: 701-732.
• Camargo EP 1964. Growth and differentiation of Trypanosoma cruzi I. Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop Săo Paulo 6: 93-100.
• Chung S-H, Gillespie RD, Swindle J 1994. Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements. Mol Biochem Parasitol 63: 95-107.
• Cooper R, Ribeiro de Jesus A, Cross GAM 1993. Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion. J Cell Biol 122: 149-156.
• Kain SR, Adams M, Kondepudi A, Yang TT, Ward WW, Kitts P 1995. Green fluorescent protein as a reporter of gene expression and protein localization. BioTechniques 19: 650-655.
• Kelly JM, Das P, Tomás AM 1994. An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector. Mol Biochem Parasitol 65: 51-62.
• Lee MGS, Van der Ploeg LHT 1990. Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei Science 250: 1583-1587.
• McCarthy-Burke C, Taylor ZA, Buck GA 1989. Characterization of the spliced leader genes transcripts in Trypanosoma cruzi Gene 82: 177-189.
• Mondragon A, Wilkinson SR, Taylor MC, Kelly JM 1999. Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates. Parasitology 118: 461-467.
• Nozaki T, Cross GAM 1994. Functional complementation of glycoprotein 72 in a Trypanosoma cruzi glycoprotein 72 null mutant. Mol Biochem Parasitol 67: 91-102.
• Nunes LR, Carvalho MR, Shakarian AM, Buck GA 1997a. The transcription promoter of the spliced leader gene from Trypanosoma cruzi Gene 188: 157-168.
• Nunes LR, de Carvalho MR, Buck GA 1997b. Trypanosoma cruzi strains partition into two groups based on the structure and function of the spliced leader RNA and rRNA gene promoters. Mol Biochem Parasitol 86: 211-224.
• Otsu K, Donelson JE, Kirchhoff LV 1993. Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene. Mol Biochem Parasitol 57: 317-330.
• Rudenko G, Le Blancq S, Smith J, Lee MG, Rattray A , Van der Ploeg LH 1990. Procyclic acidic repetitive protein (PARP) genes located in an unusually small alpha-amanitin-resistant transcription unit: PARP promoter activity assayed by transient DNA transfection of Trypanosoma brucei Mol Cell Biol 10: 3492-3504.
• Tyler-Cross, RE, Short, SL, Floeter-Winter, LM, Buck, GA 1995. Transient expression mediated by the Trypanosoma cruzi rRNA promoter. Mol Biochem Parasit 72: 23-31.
• Vanhamme L, Pays E 1995. Control of gene expression in trypanosomes. Microbiol Rev 59: 223-240.
• Zomerdijk JCBM, Ouellette M, Ten Asbroek ALMA, Kieft R, Bommer AMM, Clayton CE, Borst P 1990. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei EMBO J 9: 2791-2801.

Publication Dates

• Publication in this collection
  02 Feb 2000
• Date of issue
  Jan 2000

History

• Accepted
  11 Nov 1999
• Received
  18 Aug 1999