Cell Line Engineering

Gene editing, a form of genetic engineering, enables the introduction of permanent and site-specific genomic DNA modifications within the genome.

An array of molecular tools exists for controlling gene expression, encompassing genome editing nucleases, transposons, episomes, siRNA, and shRNA.

Notable examples of gene-editing nucleases utilized in research include meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases (Cas).

To direct the nuclease to the target site, MNs, ZFNs, and TALENs rely on protein-DNA interactions, whereas CRISPR-Cas systems are guided by RNA-DNA interactions, which provides several advantages over ZFNs and TALENs. These advantages include straightforward design for any genomic targets, easy prediction of off-target sites, and the ability to modify multiple genomic sites simultaneously (multiplexing).

CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is a revolutionary gene-editing technology derived from bacterial immune systems. It works by utilizing a guide RNA (gRNA) to direct a molecular scissors enzyme, typically Cas9, to a specific location in the genome. Once guided to the target site, the Cas9 enzyme makes a precise double stranded break in the DNA. This double stranded break is preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA.

CRISPR technology offers unprecedented precision, efficiency, and versatility in genetic manipulation, making it a powerful tool for research, biotechnology, and potential therapeutic applications.

Transfection involves introducing nucleic acids into cells using non-viral methods, while transduction entails introducing foreign DNA into cells using viral vectors.

Lentiviral transduction is a process where genetic material is introduced into a target cell using a lentiviral vector. The lentiviral vector is engineered to carry the desired genetic payload, and once introduced into the target cell, it integrates the genetic material into the host genome. This integration enables stable, long-term expression of the introduced genes.

Lentivirus transduction is widely employed in genetic research, gene therapy, and stable cell line generation due to its ability to efficiently deliver and integrate genetic material into a broad range of cell types.