Transfection of primary mouse T cells represents a major breakthrough in addressing research areas as T cell function, activation, and signaling in in vitro systems. In this study, we show transient transfection of genes into naïve mouse T cells using nucleofection, a modified electroporation technique. Using this approach, we knocked down endogenous Smad3 by efficient delivery of Smad3 shRNA into antigen-specific naïve T cells isolated from TCR-transgenic mice. The resultant transfected T cells could be safely adoptively transferred into syngeneic recipients and were fully capable of responding to TCR-mediated activation in vivo. This protocol offers the possibility for rapid functional in vivo studies of targeted genes in primary mouse T cells.
Gene transfer technologies are a crucial tool to study gene regulation as well as for the analysis of the expression and function of proteins in T cells. Most commonly, standard cell lines are used for studies in these fields because gene transfer into these cells is easy. However, they show cancer-like growth pattern and often these cell lines have deviated from the cell type they originated from. Manipulation of gene expression in T cell lines and clones by the introduction, deletion, or mutation of specific genes, has enabled dissection of molecular requirements for T cell activation, signaling and function. However, these cultured cell lines do not represent the physiologic state of normal non-transformed T cells.
In mice and rats, in vivo modulation of gene expression by transgenesis as well as knockout and knock-down technologies has been important for unraveling of functions of specific genes and pathological processes. However, these approaches are costly and time-consuming and are particularly problematic for genes that affect T cell development [1-3] or in naïve T cell differentiation , as viable animals may not develop. To overcome these limitations, attempts have been made to engineer gene expression in mature, naïve T lymphocytes. Early, murine leukemia virus-based vectors have been used to transfect genes into in primary mouse T cells activated with mitogens, anti-CD3/anti-CD28 antibodies, or antigens [5-8], resulting in infection efficencies ranging from 10% to 20%, and 40% to 90%. There are significant limitations to this approach, as resting T cells are not permissive to infection by murine leukemia viruses. Although adenoviral vectors are widely used for gene delivery into adherent cell lines, primary cells of the lymphoid lineage are largely refractory to adenoviral transduction . Furthermore, viral transduction approaches carry the considerable disadvantages of limited transgene size, time-consuming vector construction, and viral stock generation, in addition to the biohazard risk .
Recently, non-retroviral-mediated gene delivery systems, such as electroporation have been widely adapted to transfection of primary human T lymphocytes. Electroporation has been shown to efficiently introduce DNA into activated, and even freshly isolated human T cells [11-13], with transfection efficiencies of 32% in primary human T cells . In contrast, resting mouse T cells are resistant to DNA uptake via conventional electroporation [15,16]. Years ago, Amaxa set the first milestone in overcoming this obstacle with the Nucleofector Technology. It is a major improvement of the electroporation technology. Using this new technology, it has been shown that naïve CD4 positive T cells exhibited transfection efficiency of 6-12%, resting memory CD4 positive T cells exhibited substantially higher transfection efficiency (23% to 25%), and effector cells displayed the highest transfection efficiency (35%) . Recently, the Goffinet et al study demonstrated high-level non viral gene delivery in all major classes of primary lymphocytes from rodents .
In the present study, we combined the Amaxa nucleofection technology and adoptive transfer, to successfully knock-down endogenous Smad3 protein by using Smad3 shRNA. By using the resultant transfected cells we further demonstrated that Smad3 is involved in the regulation of antigen-specific T cell responses and in the induction of T cell tolerance by costimulation blockade.