Plasmid DNA delivery remains one of the most powerful, versatile and efficient gene delivery methods for various applications ranging from protein expression to gene silencing with shRNA. Our broad spectrum reagents, TransIT-X2® Dynamic Delivery System, TransIT®-2020 Transfection Reagent and TransIT®-LT1 Transfection Reagent enable delivery to a wide range of cells with maximal cell viability. Electroporation with our Ingenio® Electroporation Solution extends this list to virtually any cell type with superior delivery efficiency using a single cost-effective and convenient solution.
siRNA and miRNA Delivery
One of the most widespread techniques for interrupting gene function is RNA interference (RNAi). Small interfering RNA (siRNA) is a molecular tool used to induce RNAi for transient downregulation of gene expression in mammalian cells. Structurally, siRNAs are double-stranded and range from 20 to 24 bp in length with two overhanging nucleotides on the 5' and 3' ends. Naturally occurring microRNAs (miRNAs) are 22 nucleotide RNAs that regulate mRNAs in animal and plant cells. As with siRNA, miRNA can be delivered to cells to regulate gene expression. Transfection experiments with siRNA often involve cytoplasmic delivery of synthetic duplex RNA molecules designed to target a specific mRNA. One of the key distinctions between siRNA and miRNA is target specificity; synthetic siRNAs are typically engineered to target a single mRNA sequence, whereas miRNAs often have multiple targets. Therefore, in contrast to siRNA delivery experiments where a single gene may be targeted with high specificity, miRNA transfection experiments can be analogous to flipping a switch in order to effectively turn entire gene regulatory networks on or off. As with most cell biology experiments aimed at modifying gene function, the success of RNAi is dependent on efficient delivery. Mirus offers three distinct reagent formulations for efficient gene knockdown in a wide range of cell types: TransIT-X2® Dynamic Delivery System, TransIT-siQUEST® Transfection Reagent and TransIT-TKO® Transfection Reagent.
CRISPR Guide RNA and Cas9 RNP Delivery
The CRISPR/Cas9 genome editing platform relies on a guide RNA to direct the Cas9 endonuclease to the target site. Guide RNA delivery can be accomplished by several means. Guide RNA can be expressed off of plasmid utilizing an RNA polymerase III promoter such as the U6 promoter, or the guide RNA can be delivered as a single or two-part synthetic oligonucleotide. Many researchers utilize two-part guide RNAs consisting of a CRISPR RNA (crRNA) which defines target specificity, and a trans-activating crRNA (tracrRNA) which remains constant from one experiment to the next. Thus, this flexible approach only requires designing the crRNA when re-targeting different genomic loci. Our TransIT-X2® Dynamic Delivery System and TransIT®-mRNA Transfection Kit efficiently deliver single or two-part gRNA oligos. TransIT-X2® Dynamic Delivery System also efficiently delivers Cas9 protein and guide RNA as a ribonucleoprotein (RNP) complex. For more information on how the CRISPR/Cas9 system works, visit our CRISPR Application Page.
mRNA transfection is an attractive alternative to DNA transfection for several reasons: Messenger RNA does not require transport to the nucleus, does not require transcription and leads to rapid gene expression following transfection. DNA-free transfection methods are also advantageous due to the fact that there is no risk of insertional mutagenesis. The rapid bursts of expression associated with mRNA delivery are often ideal in genome editing experiments where long-term expression is avoided in order to mitigate off-target effects. Our TransIT®-mRNA Transfection Kit efficiently delivers mRNA as well as longer viral RNAs.
Antisense oligonucleotides (ASOs) are routinely used for modulation of gene
expression. For example, ASOs can be designed to block the function of
miRNAs in a sequence-specific manner. Oligonucleotide delivery is also becoming increasingly important for achieving precise genomic insertions following targeted cleavage by Cas9. Our TransIT®-Oligo Transfection Reagent has been used to deliver a wide range of oligonucleotides including phosphodiester DNA, phosphothioate DNA (sDNA), phosphothioate RNA (sRNA), 2'OMe RNA, 2'OMe RNA/sDNA Chimerics, Morpholino/DNA duplexes, siRNA and miRNA.