Over 10–14 days, single resistant cells divide to form visible colonies. These colonies represent "clonal populations" derived from a single successful integration event.
: This gene encodes an enzyme that detoxifies the antibiotic G418 (Geneticin). When integrated into a cell's genome, it allows that cell to survive and replicate in medium containing G418, while non-modified cells perish.
: Derived from the mouse Pgk-1 gene, this is a "housekeeping" promoter known for its robust, constitutive activity across various mammalian cell types. It is particularly effective in ES cells, where other promoters might be silenced. pgk-neo
When scientists treat cells with antibiotics like , the drug inhibits protein synthesis, leading to cell death. However, if a cell contains the neoR gene, the enzyme modifies G418, preventing it from binding to ribosomes and allowing the cell to survive and proliferate.
💡 If your cells are dying even after transfection, check your G418 kill curve; different cell lines have wildly different sensitivity levels! If you'd like, I can help you: Draft a Methods section for a paper using PGK-Neo. Compare it to other selection markers (Puro, Hygro, Blast). Explain how to design a vector including this cassette. Which of these sounds most useful for your project? Over 10–14 days, single resistant cells divide to
While CRISPR-Cas9 has revolutionized gene editing, the need for selection remains. When inserting a large DNA sequence via Homology Directed Repair (HDR), scientists often include a PGK-Neo cassette within the donor template. This ensures that the resulting colonies are not just indels (random edits) but precise insertions of the new genetic material.
Without the PGK-Neo cassette, finding the few modified cells among millions of unmodified ones would be akin to finding a needle in a haystack. When integrated into a cell's genome, it allows
While robust, the pgk-neo system is not perfect. Understanding its quirks is essential for experimental success.
To understand why this cassette is so powerful, it is necessary to examine its two core components individually.