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Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes

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Abstract

Our laboratory has developed a series of Gateway® compatible lentiviral expression systems for constitutive and conditional gene knock-down and over-expression. For tetracycline-regulated transgenic expression, we constructed a lentiviral “DEST” plasmid (pHR-TetCMV-Dest-IRES-GFP5) containing a tetracycline-responsive minimal CMV promoter, followed by an attP site-flanked DEST cassette (for efficient cloning of cDNAs by “Gateway®” recombination cloning) and green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES).This lentiviral bicistronic plasmid allows immediate FACS identification and characterization of successfully transfected cell lines. Although this system worked well with several cDNAs, we experienced serious problems with SLA, Bam and BMF. Particularly, we cloned the cDNA for human SLA (Src–like adapter), a candidate gene in GC-induced apoptosis, into this plasmid. The resulting construct (pHR-TetCMV-SLA-IRES-GFP5) was transfected into HEK 293-T packaging cells to produce viral particles for transduction of CEM-C7H2-2C8 cells. Although the construct produced many green fluorescent colonies at the HEK 293-T and the CEM-C7H2-2C8 level, we could not detect any SLA protein with α-SLA antibody from corresponding cell lysates. In contrast, the antibody readily detected SLA in whole cell lysate of HEK 293-T cells transfected with a GST-flagged SLA construct lacking IRES-GFP. To directly address the potential role of the IRES-GFP sequence, we cloned the SLA coding region into pHR-TetCMV-Dest, a vector that differs from pHR-TetCMV-Dest-IRES-GFP5 just by the absence of the IRES-GFP cassette. The resulting pHR-TetCMV-SLA construct was used for transfection of HEK 293-T cells. Corresponding lysates were assayed with α-SLA antibody and found positive. These data, in concert with previous findings, suggest that the IRES-GFP cassette may interfere with translation of certain smaller size cDNAs (like SLA) or generate fusion proteins and entail defective virus production in an unpredictable manner.

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Abbreviations

GFP:

Green fluorescent protein

IRES:

Internal ribosomal entry site

CMV:

Cytomegalovirus

DEST:

Destination

FACS:

Fluorescence activated cell sorting

pENTR:

Entry plasmid

GC:

Glucocorticoid

cDNA:

Complementary doexyribonucleic acid

EMCV:

Encephalomyocarditis virus

HEK:

Human embryonic kidney

SLA:

SrcLike adaptor

Tet:

Tetracycline

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Acknowledgments

We thank Drs. S Geley and R Kofler for stimulating discussions; S Lobenwein for technical help. This study was supported by grants from Innsbruck Cancer Aid Society and Higher Education Commission of Pakistan.

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Authors and Affiliations

  1. Division Science & Technology, University of Education, Township Campus, Lahore, Pakistan

    Muhammad Mansha, Abrar Hussain & Muhammad Tariq

  2. Division Molecular Pathophysiology, Biocenter, Medical University, 6020, Innsbruck, Austria

    Muhammad Mansha, Christian Ploner & Anita Kofler

  3. Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan

    Muhammad Wasim

  4. Department of Zoology, Lahore College for Women University, Lahore, Pakistan

    Asma Abdul Latif

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  1. Muhammad Mansha
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  2. Muhammad Wasim
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  3. Christian Ploner
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  4. Abrar Hussain
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  5. Asma Abdul Latif
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  6. Muhammad Tariq
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  7. Anita Kofler
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Correspondence to Muhammad Mansha.

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Mansha, M., Wasim, M., Ploner, C. et al. Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes. Mol Biol Rep 39, 10227–10234 (2012). https://doi.org/10.1007/s11033-012-1898-z

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  • DOI: https://doi.org/10.1007/s11033-012-1898-z

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