Elsevier

Methods

Volume 33, Issue 2, June 2004, Pages 151-163
Methods

New non-viral method for gene transfer into primary cells

https://doi.org/10.1016/j.ymeth.2003.11.009Get rights and content

Abstract

The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy. Unfortunately, the efficient gene transfer into primary cells is still problematic. In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques. Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types. The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines. This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions. In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons.

Introduction

Gene transfer into primary cells is of essential relevance for scientific and therapeutical applications like functional genomics, drug development, and gene-based medicine. Although a variety of methods for gene transfer into mammalian cells exist, they do not allow efficient and safe transfer of DNA or RNA into most primary cells. Here, we will discuss a new strategy for non-viral gene transfer into hard-to-transfect primary cells like primary B-CLL (B cell Chronic Lymphocytic Leukemia) cells, CD34+ cells, lymphocytes, cardiomyocytes, chondrocytes, and neurons. Primary cells have so far been addressed by different gene transfer strategies.

Recombinant vectors derived from viruses are used most frequently in gene transfer experiments into primary cells. Viral vectors have the advantage of high transduction efficiencies, as compared to non-viral methods.

At the same time, these methods suffer from several limitations such as the time-consuming and laborious production of vectors, elevated laboratory costs due to the high level of safety requirements, limitation of insert size, and possible immunogenic reaction in clinical human trials [1]. In summary, viral vectors currently applied in clinical research do not meet the demands for a safe gene transfer.

Non-viral gene transfer methods fall into two main categories; physical and chemical [2], [3]. The physical methods include:

  • Electroporation (areas of cell membrane break down through the electric pulse and DNA enters the cell cytoplasm).

  • Ballistic gene transfer (introduces particles coated with DNA into cells).

  • Microinjection (DNA transfer through microcapillaries into cells).

The major obstacle in these methods is the low transfection efficiency of primary cells, high cell mortality in the case of electroporation and ballistic gene transfer, and the extremely low number of transfected cells in the case of microinjection.

The major chemical method is:

  • Lipofection (negatively charged DNA molecules bind to cationic lipid particles by electrostatic interaction. The DNA–lipid complex enters the cell through endocytosis/pinocytosis).

This method also has several disadvantages, e.g., low transfection efficiency in suspension cells, and dependence on cell division as well as on high rate of endocytosis.

Section snippets

The Nucleofector technology

The Nucleofector technology is the first highly efficient non-viral gene transfer method for most primary cells and for hard-to-transfect cell lines [4], [5], [6]. This technology is based on the long-known method of electroporation, which has now been significantly improved. Cell-type specific combinations of electrical current and solutions make the technology unique in its ability to transfer polyanionic macromolecules directly into the nucleus. Thus, cells with limited potential to divide,

Gene transfer into human hematologic cells

The cellular components of the blood ultimately originate from the same progenitor cells, the hematopoietic stem cells (HSC, CD34+) in the bone marrow. These cells differentiate into the diverse cell types found in the blood, such as the lymphoid and myeloid cells of the immune system, red blood cells, and platelets.

All these cells are of great importance for immunology and hematology basic research and for the development of new therapeutic strategies [7].

Up to now the use of genetically

Conclusions

Efficient gene transfer into primary cells has been a challenge for quite some time. Available viral methods are complex and labor intensive, whereas non-viral techniques usually fail to transfect primary cells efficiently. We have demonstrated here with some examples that the Nucleofector technology represents a system that allows efficient and rapid transfection of a broad range of primary cells from different species in a reproducible manner. Furthermore, the data shown also suggest that the

Acknowledgements

The CLL experiments were supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 502 (T4). Furthermore we thank Mologen (Berlin, Germany), Axel Polack, and Georg Bornkamm (GSF, Munich, Germany) for kindly providing expression vectors.

The CH1 antibody developed by Jim Jung-Ching Lin was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. Department of Biological Sciences, Iowa City, IA 52242.

References (56)

  • C.M Wendtner et al.

    Exp. Hematol.

    (2003)
  • M Buschle et al.

    J. Immunol. Methods

    (1990)
  • F Schakowski et al.

    Mol. Ther.

    (2001)
  • W.G Wierda et al.

    Blood

    (2000)
  • D.L Hunton et al.

    J. Biol. Chem.

    (2002)
  • V.M Baragi et al.

    Osteoarthritis Cartilage

    (1997)
  • P.J Doherty et al.

    Osteoarthritis Cartilage

    (1998)
  • R Kang et al.

    Osteoarthritis Cartilage

    (1997)
  • F Hirschmann et al.

    Osteoarthritis Cartilage

    (2002)
  • R.S Goomer et al.

    Osteoarthritis Cartilage

    (2001)
  • M Barkats et al.

    Prog. Neurobiol.

    (1998)
  • M Simonato et al.

    Trends Neurosci.

    (2000)
  • P Washbourne et al.

    Curr. Opin. Neurobiol.

    (2002)
  • C.E Thomas et al.

    Nat. Rev. Genet.

    (2003)
  • S Li et al.

    Curr. Gene Ther.

    (2001)
  • T Niidome et al.

    Gene Ther.

    (2002)
  • A Hamm et al.

    Tissue Eng.

    (2002)
  • J Harriague et al.

    Nat. Immunol.

    (2002)
  • A Zernecke et al.

    Faseb J.

    (2003)
  • T.N Schumacher

    Nat. Rev. Immunol.

    (2002)
  • P Buttgereit et al.

    J. Hematother. Stem Cell Res.

    (2002)
  • A Fischer

    Cell Mol. Biol.

    (2001)
  • M.J Keating

    Semin. Oncol.

    (1999)
  • K Kuhlcke et al.

    Bone Marrow Transplant

    (2000)
  • F Sakurai et al.

    Gene Ther.

    (2003)
  • V.F Van Tendeloo et al.

    Gene Ther.

    (2000)
  • M.H Wu et al.

    Gene Ther.

    (2001)
  • A Polack et al.

    EMBO J.

    (1993)
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