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· Why Cell Type-Specific Avalanche® Transfection Reagents?
· How Were Cell Type-Specific Avalanche™ Transfection Reagents Developed?
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How Were Cell Type-Specific Avalanche™ Transfection Reagents Developed?

Four major steps:

  1. Design and produce a variety of candidate ingredients and formulations based on the latest transfection theories and combinatorial chemistry.
  2. Empirical transfection screening/confirmation
  3. Repeat from step 1 to step 2 to get the optimized formulation candidates for each specific cell type or cell line.
  4. Finalize the formulations and protocols

Step 1: New ingredient development

  • Our R&D department is a group of scientists who specialize in cell biology, molecular biology, lipid chemistry, biophysics, cell dynamics, cell metabolisms, synthetic chemistry, as well as combinatorial chemistry.
  • We are familiar with the technical specifications of all of the major commercial transfection reagents. We know their ingredients, as well as their pros and cons. All major transfection reagents fall into the following categories:
    • Different types of cationic lipids (different number and length of hydrophobic chains, as well as different modification on those hydrophobic chain, such as modifications with cholesterol and fluorine.
    • Different types of cationic polymers (different backbones, different length, branch/linear situation
    • Cationic lipids or polymers may have ester bond to make them more biodegradable.
    • Cationic lipids or polymers may have different quantities and ratio of primary, secondary, tertiary amino groups, and hydroxyl group, etc.
    • Peptides (cationic peptides, receptor ligands etc);
    • Dendrimers (activated and non-activated)
    • Calcium phosphate and other cationic molecules
  • Our knowledge and experience told us that biodegradability of ingredients in transfection reagent formulations plays a major role in reducing the toxicity as well as increasing transfection efficiency of a transfection reagent.
  • Based on the above information, knowledge, and experiences, and by using combinatorial chemistry and synthetic chemistry, we developed thousands of new ingredients, most of them are biodegradable, with a wide variety of structures variations mentioned above that theoretically would cover the need for the optimal transfection on all different type of cells. For example, different ratio and quantities of primary, secondary, and tertiary amino groups will make cationic lipids or cationic polymers with different ability to complex with nucleic acids, and form different sizes and shapes of nanoparticle complexes with different amount positive charges on the surfaces, and with different proton buffering capabilities.

Step 2: Empirical screening/confirmation

Using our high-throughput transfection screening technology, we evaluated those thousands of new ingredients, as well as compounds in some pre-existed compound libraries, and chose those that have relatively higher transfection efficiency and lower toxicity in several commonly used cell lines, sensitive cell lines, and primary cells as effective ingredients.

Step 3: Make new hybrid ingredients and formulations, and evaluate again.

  • Many of the above effective ingredients work in totally different mechanisms during the process of transfection. Like many ingredients in some commercial transfection reagents, each of the above effective ingredients has its advantages and disadvantages. We believed that some of them might synergistically work together to promote transfection. Because of this, we developed comprehensive strategies to chemically link different effective ingredients together, thus creating a series of new hybrid ingredients that might keep the advantages of each of the constituent ingredients and hide the disadvantages, and perform better than any of the individual constituent ingredients. The hybrid ingredients were evaluated again using our high-throughput transfection screening technology, and the more effective hybrid ingredients were identified.
  • Once the effective ingredients and the hybrid ingredients were identified, we worked to create the ideal formulations to enhance the transfection efficiency. Similar to the chemical linking of different ingredients, mixing different ingredients of different mechanisms together in different amounts and ratios is also an effective way to achieve the synergistic effect of different ingredients on transfection. Approximately 500 effective formulations were made from the above effective ingredients and hybrid ingredients.
  • Next, the effective formulations were screened on several representative cell types, including, but not limited to endothelial, fibroblast, and epithelial cells and lymphocyte. 172 candidate formulations that have better transfection efficiencies on all or part of the representative cell types were then identified.
  • We performed high throughput transfection screening of those 172 candidate formulations on each major type of primary cells and cell lines in comparison with 4 of the most popular commercial transfection reagents for the purpose of identifying the best candidate formulations for each specific cell type or cell line (data graphs from this screening are shown in the data section of each respective cell type/cell line specific transfection reagent).

Step 4: Finalize the final formulation and protocol

  • The few best candidate formulations were further improved with ingredients, such as ligands that specifically bind to some receptors on the surface of cell membrane, or peptide with sequence that target to the nucleus to help the DNA move into the cells and nucleus, or some ingredients that simply have fusogenic function to help the DNA to move into the cells or nucleus. With the above combined effort, one final formulation that showed the optimal balance of potency & low cytotoxicity (the low percentage of 7AAD positive cells in flow cytometry analysis) among those other best candidate formulations was chosen for each of the cell line or primary cells, and was named as the respective cell type Avalanche® Transfection Reagent.
  • In the last step, we optimized the protocols for each cell type specific transfection reagents, such as the cell seeding density, the amount of each formulation, the ratio of the formulation/nucleic acid, and other details in order to maximize efficiency/viability, as well as conveniences.

Now, with the combined efforts of our scientists at EZ Biosystems, a comprehensive series of Cell type/Cell line Specific Avalanche® Transfection Reagents for about 180 cell lines and primary cells are available. The number is still growing. Our goal is to ensure  that scientists of life sciences always have access to the right transfection reagents with the highest possible transfection efficiency and the lowest toxicity for any of their primary cells or cell lines.

Discover the appropriate Avalanche® transfection reagents for your specific primary cells or cell lines by clicking HERE.

To know why we develop Cell Type-Specific Avalanche® Transfection Reagents, click HERE.

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