Transfection is a widely used laboratory cell culture technique to introduce exogenous nucleic acids into cells. Therefore, it is often used to study gene functions and gene products in cells. Today, with the advancement of life science technology, transfection has made it possible to transfect various types of nucleic acids into mammalian cells, including: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and small non-coding RNAs such as siRNA, shRNA and miRNA. The choice of the optimal transfection method depends on factors such as cell type and source, and the form of nucleic acid introduced. There are a variety of strategies for introducing nucleic acids into cells that utilize a variety of biological, chemical and physical methods. We divide the customized transfection kits into viral vector transfection kits and non-viral vector transfection kits according to the different transfection vectors used.

Figure 1. The proposed mechanism of chemical transfection. (Fus-Kujawa A, et al.;2021)

The TransfectionVectors We Can Customize Include:

Viral Transfection Vectors

Adenovirus is a double-stranded DNA virus that has been used for gene delivery. This group of viruses can infect a wide range of dividing and non-dividing cells. Typically, adenovirus vectors are derived from human adenovirus serotypes 2 and 5. Adenovirus infection begins with the attachment of cell surface receptors and the interaction of pontoons with αvβ3 and αvβ5 integrins. Subsequently, through receptor-mediated endocytosis, the adenovirus escapes from the endosome and travels to the nucleus, where viral transcription and replication occur. After infection, adenoviral DNA does not integrate into the host cell chromosome. With this in mind, this method is safe but unlikely to induce prolonged protein expression. The expression levels of the introduced genes were initially very high, but they rapidly diminished within a few weeks. In addition, adenoviruses can be amplified at high titers and remain stable in long-term storage. However, this approach has a major disadvantage that adenoviruses tend to induce strong host immune responses when used in vivo. adeno-associated virus Adeno-associated or adeno-associated virus (AAV) is a small, single-stranded DNA, non-enveloped and replication-defective virus belonging to the Parvoviridae family. Due to its properties, adeno-associated virus (AAV) replication relies on co-infection with other viruses, mainly adenoviruses. AAV has been shown to be stable over a wide temperature and pH range with little loss of activity. Adeno-associated viruses have been shown to be less immunogenic than other viruses such as adenoviruses, however, the delivered capsid protein and nucleic acid sequences can elicit an immune response. This may lead to the development of immune memory, which may reduce the clinical efficacy of subsequent reinfection with AAV Retroviral Retroviridae are a group of RNA viruses that reverse-transcribe their genomes into DNA by enzymes commonly called reverse transcriptases (mRNA-dependent DNA polymerases). The reverse transcribed DNA can be integrated into the genome of the host cell along with existing enhancers and other regulatory elements that regulate viral gene expression. All retroviruses may be used as vectors, however, researchers have focused on four groups of vectors: lentivirus (human immunodeficiency virus, HIV), gamma retrovirus (murine leukemia virus, MLV), foamy virus (human foamy virus, HFV) and alpha retroviruses.

Non-Viral Transfection Vector

• Cationic Liposomes

This technique uses positively charged (cationic) lipids/liposomes, which are amphiphilic molecules that electrostatically interact with negatively charged (anionic) phosphate residues of DNA and cell membranes. The mechanism by which cationic lipid and nucleic acid complexes are introduced into liposomes is explained by endocytosis, followed by release of the complexes into the cytoplasm. In the case of DNA, it needs to be transported into the nucleus, while mRNA is directed and retained in the cytoplasm. Transfection of cells with cationic lipids is advantageous because of the wide range of cell types used, from primary cells to various adherent or suspension cultured cell lines, and the high efficiency of this method. It allows delivery of DNA, RNA and proteins of various molecular weights into cells and is used for transient and stable transfection.

• Cyclodextrin

Cyclodextrin, as a commonly used pharmaceutical excipient, has the characteristics of good biocompatibility and easy functional modification, so it has the potential to become an excellent gene delivery carrier. Since the introduction of cyclodextrin can reduce the cytotoxicity of other gene carriers, it has received great attention in the design of gene carrier systems. Cyclodextrin derivatives can be used directly as gene delivery vehicles, or as linkers or modifiers for the construction of other gene delivery vehicles, and can also be used for gene delivery in the form of quasi-polyrotaxane or polyrotaxane.

• Cationic Polymers (linear polymers, star polymers and dendrimers)

Many types of polymers have been successfully tested for gene delivery and/or reproductive medicine. One of the most common transfection methods is the use of cationic polymers, which have been shown to be gene delivery vehicles with high solubility in aqueous solutions. Due to their positive charge, they are an alternative to viral vectors for the introduction of pDNA, mRNA or siRNA into cells. DNA, in turn, is negatively charged due to the presence of phosphate residues in its structure, thus, it forms complexes with cationic polymers based on electrostatic interactions. The complexed pDNA protects against cellular nuclease degradation. Compared with cationic lipids, cationic polymers have more efficient DNA condensation capacity. The chemical structure of these macromolecules may be linear or branched. The group of branched polymers includes star-shaped macromolecules, and their application in gene delivery systems has received increasing attention, mainly due to the availability of efficient delivery mechanisms. The unique star structure results in high charge density. This is an advantage during interaction with genetic material.

Nanoparticles

• Polymer Nanoparticles

A variety of natural and synthetic polymers can deliver nucleic acids in an effective manner. Efficient and targeted delivery of antisense oligonucleotides (asODNs) has been demonstrated using folic acid (FA)-conjugated hydroxypropyl-chitosan (HPCS) nanoparticles (NPs). Interestingly, siRNA delivery was enhanced by surface modification of poly(lactide-glycolide) PLGA NPs with acetyl derivatives.

• Nanoparticles Made of Solid Lipids

Solid lipid nanoparticles (SLNPs) can target the delivery of therapeutic agents to specific cells. And through mannan-based PE grafting ligand modification, on the one hand, it can provide higher gene expression of SLNP in vivo, and on the other hand, it can also provide targeting opportunities for in vivo delivery.

• Inorganic Nanoparticles

It has been demonstrated that organically modified silica (ORMOSIL) NPs can achieve efficient nucleic acid delivery in vivo. The organically modified silica allows surface functionalization of NPs with amino groups of DNA, which in turn improves the monodispersity and stability of the complexes in aqueous solution. ORMOSIL, as a non-viral platform for gene delivery, has great potential for effective therapeutic manipulation of these cells.

Our Services:

• Custom Polymers In Vivo Transfection Kit Services
• Custom Cyclodextrin In Vivo Transfection Kit Services
• Custom Liposomes In Vivo Transfection Kit Services
• Custom Nanoparticles In Vivo Transfection Kit Services

Our Advantage:

• Customized transfection kits based on customer needs
• Provide a variety of delivery vehicles for users to choose
• Different vector screening can be provided to determine the optimal transfection vector
• Professional design and service team to provide you with reliable service and technical support
• Timely feedback of technical reports

CD BioSciences focuses on the development of transfection systems and customizing transfection reagents for gene transfection using our core technology. Through our high-quality products and services, the effectiveness of your transfections can be greatly improved.

Reference

1. Fus-Kujawa A, et al.; An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro. Front Bioeng Biotechnol. 2021, 9:701031.

* For research use only. Not for use in clinical diagnosis or treatment of humans or animals.