Cambio - Excellence in Molecular Biology

Transfection-EZ Biosystems

Transfection-EZ Biosystems: Why cell-type specific?

Avalanche® Transfection Reagent Series: Built on their expert knowledge & experience, powered by their vision & passion, EZ Biosystems™ has been dedicated to the research and development of next-generation transfection reagents by applying their expertise in combinatorial chemistry, molecular biology, and cell biology. With the launch of Avalanche® Transfection Reagent series, EZ Biosystems™ has been at the leading forefront of next-generation transfection reagent development and production. Check out the general purpose transfection reagents for plasmids, siRNAs (plasmid + siRNA co-transfections) and CRISPR/Cas9 as well as highly specific transfection reagents that have been carefully optimised for different primary cell types and different cell lines.

Why Cell Type-Specific   Avalanche™ Transfection Reagents?

The problems of current transfection reagents:

  • Low transfection efficiency: most transfection reagents work well on only a few commonly used cell types, such as 293T cells, Hela cells, or CHO cells. In other words, they don’t work well on those uncommon cells, most primary cells, and most suspension culture cells.
  • High toxicity: The chemical ingredients of most commercial transfection reagents usually cause too much stress to those sensitive cells, such as stem cells or primary cells. This is also one of the major reasons that caused low transfection efficiency in those sensitive cells.

Why do these problems exist?

  • Transfection is a complicated process. Although scientists understand quite a bit of transfection and gene expression processes as well as many signal pathways, there are still many aspects in the transfection process that remain unknown.
  • Cells are not created equal. Different type of cells respond differently to a certain transfection reagent. The following are some of the cell type specific factors that may have impact on transfection:     
    • The type and quantity of receptors on the cell membrane
    • lipid types and % of each type of lipids on the cell membrane
    • Glycosaminoglycans (GAG) types and quantity on the cell membrane
    • The total negative charges on the surface of the cells
    • The endocytotic ability of different type of cells
    • The types and quantity of enzymes in endosomes/lysosomes and in the cytoplasm
    • Cell proliferation ability and status
    • Other unknown properties of cells that cause the differences on their responses to transfection process.
  • Properties of a transfection reagent that may affect transfection efficiency, such as:
    • The ability of the reagent to compact the nucleic acids into nanoparticle complexes of the right sizes and shapes for a certain type of cells.
    • The correct amount of positive charges on the surfaces of the compacted nanoparticle complexes (Zeta Potential) for a certain type of cells.
    • The efficiency of the nanoparticle complexes in binding the cell surfaces
    • The efficiency for the nanoparticle complexes to stimulate the endocytosis of the cells.
    • The ability of the transfection reagent to protect the nucleic acids against the enzymes in the endosomes/lysosomes and in the cytoplasm.
    • The ability of the transfection reagent to efficiently cause the endosomal escape of the nucleic acids into the cytoplasm. Endosomal escape is the bottleneck of transfection process. In regular transfection only 1-2% of nucleic acids in the endosomes successfully escaped into the cytoplasm.
    • How efficient will the transfection reagent help to transport the nucleic acids into nucleus (for plasmid DNA)
    • The biodegradability of ingredients used in the reagent. Less biodegradability will cause cell stress, thus the toxicity issue after transfection.
    • Other unknown factors of transfection reagents that could affect transfection efficiency.
  • Even with the right transfection reagent, the protocol used on a specific type of cells also needs to be optimized in order to achieve a maximum level of transfection efficiency and a low level of toxicity.            

The best solutions: Cell type/cell line specific Avalanche® transfection reagents

Our Cell type/cell line specific Avalanche® transfection reagents are a series of transfection reagents that were derived from a large scale transfection screening and optimization process from a large pool of specifically designed, biodegradable ingredients and formulations with a wide variety of structures that theoretically cover the need for the optimal transfection on all different type of cells. For example, the pool of our cationic lipids have different number and length of hydrophobic chains, different modification status of the hydrophobic chain, such as modifications with cholesterol or fluorine etc. The pool of our cationic polymers contains different backbones, different length, different branch/linear ratio. Both cationic lipids and cationic polymers contain different quantities and ratio of primary, secondary, and tertiary amino groups, and hydroxyl group, etc.

  • After complexing with nucleic acids, they form different sizes and shapes of nanoparticle complexes with different amount positive charges on their surfaces, and with different proton buffering capabilities. Thus, as a result of the large scale transfection screening, each of these reagents, after combining with nucleic acids, is able to form nanoparticle complexes with the right sizes, shapes, the right amount of positive charges on their surface, and with the right proton buffering capabilities for the respective type of cells. Thus, the formed nanoparticle complexes are able to efficiently bind to the membrane of the respective type of cell, and effectively trigger the endocytosis process of the cells. Once the nanoparticle complexes moved into the cells through endocytosis, the nanoparticles are able to protect the nucleic acids from any enzymes in the endosomes/lysosomes due to the enclosure and tight binding between the cationic lipids/polymers and the nucleic acids. Most important, each of the reagent, due to its right proton buffering capability, is able to efficiently promote the endosomal escape of the nucleic acids from endosome/lysosome as a result of proton sponge-caused endosomal rupture at the right time and right location for easy and fast transport into the nucleus.
  • In addition, all the ingredients used are biodegradable in the cells, thus minimizing the toxicity of each formulations.
  • Together with optimized protocols for each type of cell lines or primary cells, our Cell type/cell line specific Avalanche® Transfection Reagents ensure the highest levels of transfection efficiency while keeping the toxicity levels minimal on the respective cell type.