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Efficient nonviral gene delivery into primary lymphocytes from rats and mice

Department of Virology, University of Heidelberg, Heidelberg, Germany

¹Correspondence: Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany. E-mail: oliver_keppler{at}med.uni-heidelberg.de

SPECIFIC AIMS

A rapid method for efficient nonviral gene delivery into primary rodent lymphocytes would greatly facilitate the study of signaling and metabolic pathways in untransformed hematopoietic cells as well as the validation of gene expression and targeting strategies. In the present study, we sought to develop species-adapted, nonviral gene delivery protocols for primary lymphocytes from rats and mice based on the recently introduced nucleofection technology and demonstrate their feasibility for functional downstream applications.

PRINCIPAL FINDINGS

1. Nucleofection allows high efficiency transfection of proliferating primary rat T lymphocytes
A conditioned and supplement-enriched culture medium combined with an activation protocol using concanavalin A (ConA) and human recombinant interleukin-2 (IL-2) was found to be optimal for proliferation, viability, and transfectability of cells. The rodent-adapted nucleofection protocol is based on the protocols and reagents established for human T cells by Amaxa Biosystems, but our protocol contains substantial changes concerning cultivation conditions and electrical parameters to achieve maximum transfection efficiencies (9-fold higher viability and 34-fold enhancement of transfection efficiency compared with the standard protocol; (Fig. 1 H). In a side-by-side comparison of several transfection (electroporation, nucleofection) and viral transduction (adenoviral and lentiviral) techniques, nucleofection was superior for transfection of activated rat T lymphocytes (Fig. 1) . Up to 82% transgene-positive cells (54±3%, n=15) were detected at moderate cytotoxicity. On average, a combination of the species-adapted nucleofection procedure and optimized cultivation conditions increased the transfection efficiency 10-fold over published protocols for a standard pEGFP expression plasmid and up to 370-fold for a larger, 14 kb HIV-EGFP reporter plasmid.

View larger version (59K): [in this window] [in a new window]   Figure 1. Comparative analysis of transgene delivery by transduction or transfection methods in activated primary rat T lymphocytes. Rat splenocytes were activated with ConA/IL-2 for 3 days. Cultures that had been electroporated, nucleofected (with pEGFP-C1), or adenovirally transduced were analyzed for EGFP expression at day 1. Lentivirally transduced cells were analyzed for EGFP expression at day 3 due to slower transgene expression kinetics. A) Viable cells (>90% rCD3+ T cells) were identified by gating on the live lymphocyte population (gate R1) in the FSC/SSC plot and B) on the population of propidium iodide-negative cells (gate R2). Gate R1 excludes events representing cell debris or dead cells, which always compose a significant fraction in an activated primary lymphocyte cell culture. Gate R3 defines the GFP-positive subpopulation of gate R2. C) Phase contrast and (D) fluorescence micrographs of nucleofected primary rat T lymphocytes (38% EGFP-positive cells by flow cytometry). E) Relative % of GFP-expressing viable cells (gate R3) relative to all viable cells (gate R2). F) MFI of all viable, EGFP-positive cells. Data are presented as duplicate or triplicate values from individual animals (filled triangles, n=2–15) and as the arithmetic mean (open circle) + SE of all splenocyte samples analyzed. G) Transfection index (product of % of transfected cells and MFI) is depicted for primary rat T cells transfected with pEGFP-C1 (pEGFP) or pHIV-NL4.3E–R–GFP (pHIV-EGFP) by either electroporation or nucleofection. Bars = arithmetic means +SE of values obtained from different animals (n=5). H) Transfection index of rat T cells cultivated and nucleofected according to Amaxa Biosystems’ instructions in the Human T Cell Nucleofector Kit (standard protocol) or our optimized cultivation and nucleofection protocol. Respective electrical programs are in brackets.

2. Reduction of surface-exposed CD4 by an endocytosis-inducing viral protein or by small interfering RNA oligonucleotides
The high transfection efficiency achieved by the nucleofection procedure allowed for rapid functional ex vivo analyses of two different effectors of cell surface levels of the human CD4 receptor expressed on primary T cells from transgenic rats. We first explored whether the transient expression of the Nef protein of human immunodeficiency virus (HIV) induced down-regulation of the viral binding receptor human CD4 from the cell surface. In human T cells, Nef exploits a central transport pathway of eukaryotic cells by triggering CD4 endocytosis and subsequent lysosomal degradation. We had generated transgenic rats that coexpress human CD4 (hCD4) and CCR5 (hCCR5), the HIV entry receptor complex, on rat CD4 T cells. hCD4-transgenic rat T cells nucleofected with plasmid vectors encoding EGFP fusion proteins of HIV Nef down-regulated cell surface levels of hCD4 in a concentration- and signature motif-dependent manner. This indicated that the cellular machineries required for the endocytosis-inducing activity of the viral Nef protein are conserved in primary T cells from transgenic rats and demonstrated the integrity of these nucleofected cells for a complex biological activity.

Second, we investigated whether primary rat T cells are accessible to RNA interference technology upon nucleofection. With cell surface-exposed hCD4 again as a target, we used a small interfering RNA (siRNA) specific for hCD4 (hCD4-siRNA) to establish a gene-specific knockdown. Primary rat lymphocyte cultures nucleofected with hCD4-siRNA oligonucleotides exhibited a 50% decrease in expression of surface-hCD4 relative to control siRNA cultures at day 3 post-transfection. These results demonstrated the feasibility and specificity of RNA interference in primary rat T cells with the nucleofection technology.

3. Efficient nucleofection of activated primary mouse lymphocytes
We explored whether primary mouse lymphocytes from two widely used inbred mouse strains can also be transfected efficiently by nucleofection. Nucleofection of spleen-derived T lymphocytes from mice from strains C57BL/6- and BALB/c yielded transfection efficiencies of up to 28 and 55%, respectively. Unlike rat T cells, primary mouse T cells allowed for adenoviral and lentiviral transduction at reasonable gene delivery efficiencies (i.e., 8% for C57BL/6- and 6–19% for BALB/c-derived cells). In contrast to viral transduction, nucleofected cells showed higher levels of EGFP expression with MFI values enhanced 7-fold.

4. Activated B cells and natural killer cells are targets of nucleofection
To better define which types and subtypes of cells from the lymphoid lineage in rodents can be transfected by nucleofection, splenocyte cultures were supplemented with dextran sulfate, lipopolysaccharide, or ConA/IL-2 to promote lineage- and species-specific survival and proliferation. Nucleofected cultures were first characterized by lineage-specific markers; subsequently these lymphocyte subpopulations were analyzed for transgene expression levels. Remarkably, significant percentages of cells from all three major lymphocyte subpopulations derived from rat or mouse splenocytes [i.e., B cells (8–17%), natural killer (NK) cells (19–38%)], CD4 T cells (39%), and CD8 T cells (28%), expressed high transgene levels (Fig. 2 ). Thus, nucleofection is the first technology that renders primary B cells and NK cells from both rats and mice accessible to efficient nonviral gene delivery.

View larger version (62K): [in this window] [in a new window]   Figure 2. All major lymphocyte subtypes can be transfected by nucleofection. After lineage-specific activation, splenocytes from A–F) 3–4 Sprague-Dawley rats or F) 3–4 C57BL/6 mice were nucleofected with either A) an empty vector for autofluorescence compensation or B–E) an EGFP-encoding vector and assessed for EGFP expression 8 h later. A) Transfected, viable, 7-AAD-negative lymphocyte subsets were identified (B–E, left panels) by subset-specific markers, then (B–E, right panels) the relative % of EGFP-expressing cells were determined. F) Transfection efficiencies (arithmetic mean +SE) from duplicate samples of splenocytes from 3–4 animals are shown.

CONCLUSIONS AND SIGNIFICANCE

Functional ex vivo analyses of transgenes in primary rodent lymphocytes have been severely hampered by the lack of an efficient and rapid gene delivery method. To our knowledge, this is the first study to systematically address and successfully demonstrate high-level nonviral gene delivery in all major classes of primary lymphocytes from rodents. For most immunological studies, mice and rats are the prime model organisms. Rats are now receiving growing attention as experimental model organisms in diverse research areas including genetics, immunology, physiology, infectious diseases, and behavioral sciences as highlighted in a recent special issue on rats in Nature.

In a side-by-side comparison, species-adapted nucleofection procedures were 10- to 370-fold more effective than other nonviral and viral gene delivery methods. This was particularly striking for proliferating rat T cells, for which transfection efficiencies of > 80% could be achieved (Fig. 1) . Remarkably, cells from all three major lymphoid lineages could be transfected by the same nucleofection procedure. After cell type-specific stimulation and nucleofection, 8–39% of B cells and NK cells expressed the reporter transgene at relatively high levels (Fig. 2) . So far, primary B and NK cells have been considered virtually refractory to nonviral gene delivery.

We found that siRNA oligonucleotides can be delivered with high efficiency into activated primary rat T cells. The RNA interference technology allows the manipulation of expression levels of specific proteins and holds promise as a tool for validating drug targets and treating diseases. In primary rodent lymphocytes, the efficient delivery of siRNA could provide a rapid and simple prevalidation strategy for in vivo knockdown or knockout approaches (Fig. 3 ). Applying a target-specific siRNA, cellular CD4 levels were reduced by 50% on lymphocyte bulk cultures. The functional integrity of nucleofected rodent lymphocytes was also demonstrated by the specific down-modulation of surface-exposed CD4 after transient expression of the endocytosis-inducing HIV-1 Nef protein.

View larger version (42K): [in this window] [in a new window]   Figure 3. Schematic diagram.

Nucleofection, like other nonviral gene delivery methods, has several advantages over viral vectors, including easy and quick handling, low or no immunogenicity (and thus potential for repeated administration), and no major constraints for transgene size.

We identify the nucleofection technology as a universally applicable nonviral gene delivery method for plasmid DNA and siRNA oligonucleotides in activated rodent lymphocytes. Our species-adapted nucleofection protocols make all three major primary lymphocyte populations (i.e., B cells, NK cells, and T cells) accessible to genetic manipulation. For research areas including immunobiology, immunogenetics, immunobiochemistry, and infectious diseases, the results and methods presented in this manuscript represent a methodological breakthrough in addressing questions on lymphocyte function, activation, regulation, and signaling. The presented results and methods may contribute to the advancement of gene therapy approaches.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4651fje;

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