Transient expression of green fluorescent protein in integrase-defective lentiviral vector-transduced 293T cell line

Fazlina Nordin, Zariyantey Abd Hamid, Lucas Chan, Farzin Farzaneh, M. K Azaham A Hamid

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Citations (Scopus)

Abstract

Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors. Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fl uorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fl uorescence microscope and FACS analysis. Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells. Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages159-173
Number of pages15
Volume1448
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1448
ISSN (Print)10643745

Fingerprint

Integrases
HEK293 Cells
Green Fluorescent Proteins
Spleen Focus-Forming Viruses
Cell Line
Proteins
Transgenes
U937 Cells
Insertional Mutagenesis
Mutation
Catalytic Domain
Genome
Lentivirus
Replication Origin
Enzymes
Reporter Genes
Genetic Therapy
Monocytes

Keywords

  • D64 point mutation
  • GFP reporter gene
  • Integrase-defective lentiviral vector
  • Transduction efficiency
  • U937 cell lines

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Biology
  • Genetics

Cite this

Nordin, F., Abd Hamid, Z., Chan, L., Farzaneh, F., & Hamid, M. K. A. A. (2016). Transient expression of green fluorescent protein in integrase-defective lentiviral vector-transduced 293T cell line. In Methods in Molecular Biology (Vol. 1448, pp. 159-173). (Methods in Molecular Biology; Vol. 1448). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3753-0_12

Transient expression of green fluorescent protein in integrase-defective lentiviral vector-transduced 293T cell line. / Nordin, Fazlina; Abd Hamid, Zariyantey; Chan, Lucas; Farzaneh, Farzin; Hamid, M. K Azaham A.

Methods in Molecular Biology. Vol. 1448 Humana Press Inc., 2016. p. 159-173 (Methods in Molecular Biology; Vol. 1448).

Research output: Chapter in Book/Report/Conference proceedingChapter

Nordin, F, Abd Hamid, Z, Chan, L, Farzaneh, F & Hamid, MKAA 2016, Transient expression of green fluorescent protein in integrase-defective lentiviral vector-transduced 293T cell line. in Methods in Molecular Biology. vol. 1448, Methods in Molecular Biology, vol. 1448, Humana Press Inc., pp. 159-173. https://doi.org/10.1007/978-1-4939-3753-0_12
Nordin F, Abd Hamid Z, Chan L, Farzaneh F, Hamid MKAA. Transient expression of green fluorescent protein in integrase-defective lentiviral vector-transduced 293T cell line. In Methods in Molecular Biology. Vol. 1448. Humana Press Inc. 2016. p. 159-173. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-3753-0_12
Nordin, Fazlina ; Abd Hamid, Zariyantey ; Chan, Lucas ; Farzaneh, Farzin ; Hamid, M. K Azaham A. / Transient expression of green fluorescent protein in integrase-defective lentiviral vector-transduced 293T cell line. Methods in Molecular Biology. Vol. 1448 Humana Press Inc., 2016. pp. 159-173 (Methods in Molecular Biology).
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