Neurobiology Transfection Guide

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The field of neurobiology is rapidly growing, with researchers providing new insights into a variety of areas essential for human health, including memory, mood disorders, aging, and disease. As the field moves toward more physiologically relevant models, such as primary neurons and small animal models, the need for new nucleic acid delivery tools is rapidly increasing. One of the greatest challenges is the ability to manipulate the desired cell models that are traditionally difficult to transfect, including primary neurons and neural stem cells. Methods typically used have not met these needs—reagent-mediated DNA transfection is inefficient for nuclear entry; electroporation requires cell disruption and special instrumentation, and is expensive; and virus-mediated transduction can be very time-consuming [1].

To facilitate the use of more relevant cell models, we have developed Invitrogen™ Lipofectamine™ MessengerMAX™ Transfection Reagent, which we recommend as the primary gene delivery solution (Figure 1). Here we provide an overview for how Lipofectamine MessengerMAX reagent is used for high-efficiency transfection of neuronal cell models, enabling improved results and simplified workflows for applications such as genome editing. We also compare Lipofectamine MessengerMAX reagent to other gene delivery solutions and highlight the advantages and drawbacks of each approach.

*Refer to “Lentiviral production” (page 4) for details on high-efficiency lentiviral production using Lipofectamine 3000 reagent.
Note: The numbers in the alternative transfection methods correspond to our recommended order.

Figure 1. Recommended transfection methods, by cell type. The high transfection efficiency of Lipofectamine MessengerMAX reagent makes it the preferred choice for transfection of neuronal cell models. For researchers interested in other delivery methods, we also offer Lipofectamine reagents for DNA transfection and lentiviral production.

Lipofectamine MessengerMAX reagent is an mRNA transfection reagent that offers superior transfection efficiency in primary neurons, neural stem cells, and immortalized neural cells. Transfection of mRNA only requires entry into the cell cytoplasm, not the nucleus, and therefore greatly improves transfection efficiency in post-mitotic cells or slowly dividing cells (Figure 2). This mechanism also allows mRNA transfection to be utilized for novel applications such as genome editing where genomic integration is a major concern (see Genome editing below for further discussion).

Figure 2. Faster protein expression with no risk of genomic integration. Transfection of mRNA with Lipofectamine MessengerMAX reagent typically results in faster protein expression with greater homogeneity of expression among the transfected cells. Additionally, delivery of mRNA does not require nuclear entry (step 4), which eliminates the risk of genomic integration and makes transfection efficiency cell cycle–independent.
 

Any plasmid or PCR product with the gene of interest regulated by a T7 promoter can be used as the template for mRNA preparation (Figure 3). The mMESSAGE mMACHINE™ T7 Ultra Transcription Kit (Cat. No. AM1345) is recommended for high-yield synthesis of mRNA transcripts containing a 5′ cap and poly(A) tail for transfection. Even though mRNA transfection has become a widely used approach for gene delivery in nondividing cells, some misconceptions still remain about the methods involved (Table 1).

Figure 3. Workflow for mRNA transfection using Lipofectamine MessengerMAX reagent.

Lipofectamine MessengerMAX reagent delivers outstanding transfection efficiency in neural cell models, including primary neurons, human neural stem cells (hNSCs), and immortalized neural cell lines (Figure 4). The high transfection efficiency and homogeneity of expression among transfected cells enables improved application outcomes for a broad range of experiments.

Figure 4. Transfection of neural cell models.Lipofectamine MessengerMAX reagent was used to deliver mRNA encoding GFP (500 ng/well) in primary cortical neurons (freshly isolated from E16 mouse; 5 days in vitro), SK-N-SH cells (neuroblastoma), bEnd.3 cells (endothelial polyoma from cerebral cortex), and SHSY5Y cells (neuroblastoma) in a 24-well format. For Gibco™ Human Neural Stem Cells (hNSCs), 250 ng/well of mRNA was used in a 48-well format. Results are compared to plasmid DNA delivered with the leading DNA transfection reagent. GFP expression was analyzed 24 hours posttransfection.
 

We offer a variety of other gene delivery options based on your research needs, including lentiviral delivery and physical methods such as electroporation. However, there are advantages and limitations for each method based on the cell models being used and the end goal of the experiment: gene expression or gene knockdown. An overview of our most effective delivery solutions for neurobiology research is shown in Table 2.

Table 2. Gene delivery products for neurobiology research by nucleic acid type.

If viral delivery is the preferred method for transfecting neurons, Invitrogen™ Lipofectamine™ 3000 Transfection Reagent can be used as a highly efficient tool for lentiviral production. This versatile reagent enables high viral titers even with genes that are large or difficult to package. Production of high-quality, high-titer lentivirus can be achieved while minimizing time and reagent usage. Higher viral titers are obtained with Lipofectamine 3000 reagent than with other commonly used transfection reagents, even with larger gene sizes (Figure 5). However, viruses present challenges in production and handling, making mRNA transfection an attractive alternative to lentiviral delivery.

Figure 5. Lipofectamine 3000 reagent performs significantly better than leading competitor reagents. (A) Titers obtained using Lipofectamine 3000 reagent compared to commonly used transfection reagents in 293FT cells using a 3 kb insert gene. (B) Summary of increases in titer levels using two different cell lines and insert sizes.
 

In vivo delivery
There is considerable interest in using in vivo delivery of siRNA and mRNA to better understand diseases and for research as therapeutic molecules [5,6]. Current technologies such as viral or hydrodynamic delivery are powerful but can be time-consuming and expensive. Invitrogen™ Invivofectamine™ 3.0 Reagent is an animal origin–free transfection reagent designed for in vivo delivery of siRNA and miRNA to hepatocytes following tail-vein injection. Complexes of Invivofectamine 3.0 Reagent and siRNA are prepared and delivered using a simple procedure with no measured toxicity or stress response in the animal. Recently, we have also demonstrated that Invivofectamine 3.0 Reagent can successfully deliver mRNA to the liver, spleen, muscle, and xenograft tumors, making this reagent a promising tool for gene delivery to other areas of the body, including direct injection to the brain (Figure 6) [7].

Genome editing
mRNA transfection is widely used for genome editing using the CRISPR-Cas9 system (Figure 7). The effectiveness of the CRISPR-Cas9 system is contingent upon maximum expression of the Cas9 nuclease and guide RNA (gRNA) that targets the genomic locus of interest. Precise cleavage of DNA at a specific locus allows for the knockout of a specific gene or the insertion of a new sequence using endogenous cellular repair mechanisms.

Figure 7. Lipofectamine MessengerMAX reagent enables efficient genome editing using the CRISPR-Cas9 system. (A) Mouse Neuro 2A cells were transfected with Invitrogen™ GeneArt™ CRISPR Nuclease mRNA encoding Cas9 and in vitro–transcribed (IVT) gRNA targeting the ROSA26 or NANOG locus in a 24-well plate format. Results are compared to an all-in-one plasmid expressing both Cas9 and gRNA that was transfected into cells using Lipofectamine 3000 reagent. (B) Induced pluripotent stem cells were transfected with GeneArt CRISPR Nuclease mRNA and IVT gRNA. Cells were harvested 72 hours posttransfection and genome editing efficiency was assessed using the Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit. The cleavage products are indicated with arrowheads.
 

Conclusions

As we have shown, mRNA transfection with Lipofectamine MessengerMAX reagent provides numerous benefits when working with difficult-to-transfect cell models, including ease of use and superior efficiency. This reagent is also gentle on cells and enables high cleavage efficiency when used with genome editing tools such as the CRISPR-Cas9 system. Therefore, while there are many options for transfecting neurons, we recommend Lipofectamine MessengerMAX reagent as the primary gene delivery solution.

References

1. Karra D and Dahm R (2010) Transfection techniques for neuronal cells. J Neurosci 30:6171–6177. 
2. Technical Note: Working with RNA: the basics. Avoiding, detecting, and inhibiting RNase. 
3. Zou S, Scarfo K, Nantz MH et al. (2010) Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells. Int J Pharm 389:232–243. 
4. Kariko K, Muramatsu H, Welsh FA et al. (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16:1833–40. 
5. Sahin U, Kariko K, Tureci O (2014) mRNA-based therapeutics—developing a new class of drugs. Nat Rev Drug Disc 13:759–780. 
6. Yin H, Kanasty RL, Eltoukhy AA et al. (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15:541–555. 
7. Mouri A, Sasaki A, Watanabe K et al. (2012) MAGE-D1 regulates expression of depression-like behavior through serotonin transporter ubiquitylation. J Neurosci 32:4562–4580.