Viromer® in immunology research

High-performance transfection of MPS cells using the VIROMER® technology

The mononuclear phagocyte system (MPS) is commonly defined as a super-family of different cell types comprising circulating monocytes, tissue-resident macrophages, antigen-presenting dendritic cells plus all their lineage-committing progenitors from embryonic origin or bone marrow stem cells. Ongoing research gives increasing evidence of the multiple and complex implications of these cells in health and diseases, e.g. homeostasis, support of tissue functions, control of pathogens, and induction of immune responses in infection, inflammation, wounding or malignancy. Beyond this consensus, there is confusion and debate among immunologists to classify these cells as their physiological functions, phenotypes and locations create overlaps of the different subsets.

Notwithstanding, strategies to target MPS cells are of growing importance both scientifically and therapeutically. As part of this challenge, transfection is a precious tool to induce or inhibit gene expression, transcriptional regulation and specific functions of these cells. However, MPS cells are known to be difficult targets. They are likely very efficient for scanning the extra-cellular environment and uptaking materials but have a strong ability of self-defense. Related response mechanisms prevent further processing after intracellular delivery as required by most of common transfection methods.

The Viromer® technology has addressed that challenge. Using a high-tech polymer that emulates viral uptake, efficient transfection of MPS cells was made possible. Recent publication record and user’s feedback show proven high performance for transfecting monocytes, macrophage-like cells, and dendritic cells, positioning the Viromer® reagents as optimal tools for current and future immunology research. Within the technology, Viromer® BLUE and GREEN are selective for siRNA or miRNA and Viromer® RED transfects pDNA or mRNA.

Delivery of Cy3b-tagged (magenta) GFP-encoding mRNA into human primary macrophages using Viromer® RED (fluorescence microscopy). Data courtesy of D. Russell, Cornell University, USA.

Uptake of a GFP-labeled siRNA by KG-1a human pro-myeloblasts transfected with Viromer® BLUE. J. Lung, Chang Gung Memorial Hospital, Chiayi, Taiwan

siRNA-mediated gene silencing

Up to 80-100% complete specific gene knock-downs have been achieved by transfecting siRNA into different kinds of immune cells using Viromer® BLUE  or  Viromer® GREEN:

Note: the Viromer® BLUE and Viromer® GREEN are both based on a branched PEI hugely substituted with secondary chains of fatty acids and alkyl groups. The two polymers differ in size, linearity and substitution degrees but follow the same workflow enabling a pH-sensitive escape from the endosome compartment to deliver siRNA in targeted cells. While the Viromer® BLUE has been identified as the most versatile reagent, we have noticed a better efficiency of the Viromer® GREEN for the most precursor stages among cell lineages, e.g. undifferentiated THP-1, bone-marrow or peripheral blood progenitors.

Protein expression: mRNA versus plasmid DNA transfection

Expression of proteins through plasmid DNA transfection in monocytes, macrophages or dendritic cells is limited to certain model cell lines like RAW264.7 macrophages or THP-1 monocytes, and it is not always reliable in terms of efficiency and cell viability.

It is commonly assumed that cells of the MPS are “resistant” to plasmid transfection as part of the innate immune system. Once imported in the cytosol, double-stranded DNA is recognized by intracellular receptors activating enzymatic cascades like AIM-2/IL and cGAS-STING/IF before it can reach the nucleus, then inhibiting further processing. It does not mean that transfection reagents are not able to transport DNA into the cells, but that DNA has simply no chance to reach the nucleus.

As an alternative, transfecting cells with mRNA results in effective and reliable protein expression. Upon delivery, mRNA is directly expressed in the cytosol through a promoter-independent process and protein is detectable as early as 2 or 4h post-transfection.

As shown in the table below, mRNA provides a very productive template for translation and significantly increases the transfection efficiency of the Viromer® RED reagent. This improvement is also related to current modified mRNA chemistries that have solved much of the instability associated with its native form and its capacity to elicit innate immune responses.

Comparative transfection efficiency of Viromer® RED used for plasmid DNA or mRNA delivery in diverse immune cell lines (maximal percentage of positive cells reported by users).

To compare transfection efficiency of the Viromer® technology with both cargoes, we suggest using the Viromer® ONE RED GFP Positive Controls.

Transfection of primary human macrophages with pCMV-GFP plasmid and GFP-encoding mRNA using Viromer® ONE RED GFP Controls. Expression of GFP was only observed with mRNA, no signal with plasmid DNA. Contributor not disclosed

Colors

Viromer® BLUE | Viromer® GREEN | Viromer® RED | Viromer® YELLOW

transfection efficiency

< 30% | 30-50% | 50-80% | > 80%

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Type siRNA mRNA pDNA Data
BV-2            See data Publications
CD34+ haematopoietic cells, primary human      Publication
Dendritic cells, primary human and mouse                See data Publications
Hap1    
HT      Publication
J774                See data
Kasumi-1      See data
KG-1a      See data
Macrophages, primary human and mouse                 See data Publications
Microglia, primary murine      See data Publications
Monocytes, primary human               See data Publications
MV4-11     See data
N9      See data
PBMCs, primary human      See data
Ramos     Publication
RAW 264.7              See data Publications
THP-1                 See data Publications
U-937     Publication