Fluorescent Leishmania species: Development of stable GFP expression and its application for in vitro and in vivo studies

Bolhassani, Azam; Taheri, Tahereh; Taslimi, Yasaman; Zamanilui, Soheila; Zahedifard, Farnaz; Seyed, Negar; Torkashvand, Fatemeh; Vaziri, Behrouz; Rafati, Sima

doi:10.1016/j.exppara.2010.12.006

Cited by 79 publications

(84 citation statements)

References 26 publications

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“…Pixel counting and measurement of the lesions were performed using KODAK molecular image software version 5.3. Results were reported as "net intensity", a quantitative measurement defined as the number of green pixels in a given area (region of interest) multiplied by the average intensity of each pixel [22,23]. This experiment was performed 7 weeks post challenge since earlier imaging barely discriminates fluorescent signal difference among groups.…”

Section: In Situ Imaging Of Anesthetized Mice For Egfp Fluorescencementioning

confidence: 99%

Assessment of Protection Induced by DNA and Live Vaccine Encoding Leishmania MHC Class I Restricted Epitopes against L. major Challenge in Balb/c Mice Model

Zandieh¹,

Kashi²,

Taheri³

2015

J Microb Biochem Technol

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Section: In Situ Imaging Of Anesthetized Mice For Egfp Fluorescencementioning

confidence: 99%

Assessment of Protection Induced by DNA and Live Vaccine Encoding Leishmania MHC Class I Restricted Epitopes against L. major Challenge in Balb/c Mice Model

Zandieh¹,

Kashi²,

Taheri³

2015

J Microb Biochem Technol

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“…For transfection, 4 Â 10 7 log-phase parasites re-suspended in ice-cold electroporation buffer (pH ¼ 7.5) containing 8 mg of linearized pLEXSY-E7-NT-GFP or pLEXSY-E7-CT-GFP, stored on ice for 10 min and electroporated using Bio-Rad Gene Pulser Ecell under conditions of 500 mF, 450 V and pulse time $5-6 ms. The electroporated promastigotes were plated on solid media containing 50 mg/ml of G418 (Bolhassani et al, 2011). Clones were selected on Noble agar plates and further propagated in liquid M199 10% medium in the absence of G418.…”

Section: Parasite Growth and Transfectionsmentioning

confidence: 99%

“…The L. tarentolae strain (ATCC 30267) was grown at 26 C in complete M199 medium (Bolhassani et al, 2011). For transfection, 4 Â 10 7 log-phase parasites re-suspended in ice-cold electroporation buffer (pH ¼ 7.5) containing 8 mg of linearized pLEXSY-E7-NT-GFP or pLEXSY-E7-CT-GFP, stored on ice for 10 min and electroporated using Bio-Rad Gene Pulser Ecell under conditions of 500 mF, 450 V and pulse time $5-6 ms.…”

Section: Parasite Growth and Transfectionsmentioning

confidence: 99%

“…In the present study, we have used a Leishmania expression vector (LEXSY), which can mediate high-level production of heterologous proteins both intracellularly and extracellularly (Bolhassani et al, 2011). L. tarentolae was transfected with the expression cassettes containing E7-NT (gp96) or E7-CT (gp96) genes fused to the green fluorescent protein (GFP) as a reporter gene in order to rapidly and sensitively quantify Leishmania promastigotes.…”

Section: Introductionmentioning

confidence: 99%

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A non-pathogenic live vector as an efficient delivery system in vaccine design for the prevention of HPV16 E7-overexpressing cancers

et al. 2013

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The attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT

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“…On the basis of these considerations, there is a strong need to develop appropriate screening technologies. The use of fluorescent reporter genes such as those coding for green fluorescent protein (GFP) (17)(18)(19) and the red versions red fluorescent protein (RFP) (20,21) and mCherry (21,22) have considerably facilitated the screening and testing of antimicrobial agents against axenic amastigotes and intracellular amastigotes, allowing both in vitro and real-time visualization in vivo. Although animal models are well established for large-scale primary drug screening, its practical use is limited due to high costs.…”

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confidence: 99%

In Vitro Screening Test Using Leishmania Promastigotes Stably Expressing mCherry Protein

Vacchina

Morales

2014

Antimicrob Agents Chemother

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Transgenic Leishmania major and Leishmania donovani axenic promastigotes constitutively expressing mCherry were used for in vitro antileishmanial drug screening. This method requires minimal sample manipulation and can be easily adapted to automatic drug tests, allowing primary high-throughput screenings without the need for expensive and sophisticated instruments.P rotozoan parasites of the genus Leishmania are the causative agents of a wide spectrum of human and animal diseases. The clinical manifestations of Leishmania infections range from lesions of the skin and mucous membranes to lethality, the latter caused by visceral species, including Leishmania donovani (1). Leishmania species cause morbidity and mortality throughout large areas of the Old and New World. Leishmaniasis is an emerging disease with an annual incidence of 2 million cases, and more than 12 million people are infected in over 80 countries where the disease is endemic (2), causing 80,000 deaths per year, and 350 million people are at risk of infection and disease. The increased prevalence of Leishmania-human immunodeficiency virus (HIV) coinfection is the major reason for the recent emergence of leishmaniasis in the Western world (3).Since their discovery in the 1940s, the highly toxic pentavalent antimonials [Sb(V)] have been the primary first-line treatment for all types of leishmaniasis in most parts of the world. However, the increasing frequency of relapse in leishmaniasis patients is forcing the use of other chemotherapeutic agents, including amphotericin B, isethionate pentamidine, paromomycin, and miltefosine (4, 5). Unfortunately, the majority of these antiparasitic drugs present severe side effects, there is no guarantee of cure, and some of them are frequently accompanied by emergence of drug resistance (6-8).Alternative treatments are needed; however, advances in drug discovery approaches have not been satisfactory. Although the use of axenic promastigote cultures of Leishmania as a primary screening method has being validated (7-11), the traditional approaches employed to determine the drug effect in culture present several shortcomings. Microscopic counting is prone to error, time-consuming, and requires trained personnel; the use of tetrazolium salts such as MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] to measure cell viability is labor-intensive, and results are not always reliable (12). The more sensitive and less labor-intensive colorimetric resazurin-based method may present variable metabolic behavior under different cell culture conditions. Furthermore, it is not appropriate to perform kinetic experiments due to the significant cytotoxicity that results from prolonged exposure to the reagent (13-15). Other disadvantages of these metabolic assays are the requirement to incubate the substrate with viable cells at 37°C for a sufficient time to generate a measurable signal. This may lead to undesirable artifacts resulting from interactions between the reagent, the tested drug, and the biochemistry ...

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Fluorescent Leishmania species: Development of stable GFP expression and its application for in vitro and in vivo studies

Cited by 79 publications

References 26 publications

Assessment of Protection Induced by DNA and Live Vaccine Encoding Leishmania MHC Class I Restricted Epitopes against L. major Challenge in Balb/c Mice Model

Assessment of Protection Induced by DNA and Live Vaccine Encoding Leishmania MHC Class I Restricted Epitopes against L. major Challenge in Balb/c Mice Model

A non-pathogenic live vector as an efficient delivery system in vaccine design for the prevention of HPV16 E7-overexpressing cancers

In Vitro Screening Test Using Leishmania Promastigotes Stably Expressing mCherry Protein

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