What are CHO cells and why are they significant?

The Chinese hamster ovary (CHO) cell line is a type of cell frequently used in biopharmaceutical production and basic research because it is adept at producing complex proteins that closely resemble their natural counterparts. These cells are preferred in creating essential drugs and understanding biological processes due to their capability to efficiently produce complex proteins, which is important for creating functional biopharmaceuticals.

What is the Flp-In system and why is it used?

The Flp-In system is a two-step method for inserting genes into specific locations within the CHO cell genome. First, a Flp-In cassette is integrated into the target cell line, such as DG44 or Lec 3.2.8.1 CHO cells. Then, a plasmid carrying the gene of interest (GOI) is introduced, along with the Flp-recombinase enzyme. This enzyme ensures the GOI is precisely inserted into the Flp-In cassette, offering a more reliable and efficient gene insertion method than traditional techniques. This method is important because it enhances the efficiency and precision of gene insertion, resulting in high-level and consistent protein expression.

What are Lec 3.2.8.1 and DG44 CHO cells?

Lec 3.2.8.1 CHO cells, developed by Stanley, are a specific type of CHO cell that lacks N-type glycosylation due to a mutation in their glycosylation enzymes. This lack of glycosylation results in more homogenous glycosylation of proteins, making these cells particularly useful for applications where consistent protein glycosylation is crucial, like protein crystallography. DG44 CHO cells, developed by Chasin and Urlaub, lack the enzyme DHFR, so they depend on external sources of nucleosides. This feature enables a selection process for cells that express the DHFR enzyme along with the desired gene, thereby simplifying the generation of stable cell lines.

What are the differences between the Flp-In system and other gene insertion methods?

Traditional methods like lipofectamine and electroporation introduce foreign DNA, but often integrate it into less active regions. The Flp-In system, in contrast, offers targeted insertion into specific genomic locations. Viral systems offer better integration but pose safety concerns. The Flp-In system resolves these limitations by using the Flp-recombinase enzyme to precisely insert the gene of interest into a predetermined location within the cell's genome, improving the reliability and efficiency of gene insertion, thus leading to more consistent protein expression and higher yields.

How does a fluorescent marker improve the Flp-In system?

The fluorescent marker mCherry facilitates fluorescence-activated cell sorting (FACS) to rapidly identify and isolate cells where the Flp-In cassette has been successfully integrated. This approach streamlines the selection process, making it quicker and easier to generate stable cell lines for protein production. The mCherry tag helps researchers visually pinpoint cells that have correctly incorporated the Flp-In cassette. This is a major advance in cell line development for protein production, greatly reducing the time and effort required to identify and isolate cells with the desired genetic modifications.

CHO cells producing proteins

Speed Up Protein Production: How to Create Easy-to-Use CHO Cell Lines

Theo Raines in Tech & Innovation December 2025 • 4 min read.

"Discover how a modified Flp-In system slashes the time and effort needed for constitutive protein expression in Chinese hamster ovary (CHO) cells."

The Chinese hamster ovary (CHO) cell line is a workhorse in biopharmaceutical production and a crucial tool for basic research. These cells are masters at producing complex proteins that closely resemble their natural counterparts. But getting foreign DNA into specially modified CHO cells, like DG44 and Lec 3.2.8.1, can be a real bottleneck.

Traditional methods like lipofectamine and electroporation, while common, often lead to the foreign DNA integrating into less active regions of the cell's genome. Viral systems offer better integration, but come with safety concerns and complex handling requirements. This is where the Flp-In system steps in, offering a more reliable and efficient way to insert genes into specific locations within the CHO cell genome.

Now, researchers have refined the Flp-In system, creating Flp-In-ready DG44 and Lec 3.2.8.1 CHO cell lines. By adding a fluorescent tracer protein tag (mCherry™), they've streamlined the selection process, making it faster and easier than ever to generate stable cell lines for protein production. This article dives into how this modified system works and why it matters for biopharmaceutical development and beyond.

The Power of Flp-In: Targeted Gene Insertion

The Flp-In system, introduced by Schlake and Bode, offers a two-step approach to overcome the limitations of traditional transfection methods. First, a Flp-In cassette is integrated into the target cell line. Then, a plasmid containing the gene of interest (GOI) is introduced, along with the Flp-recombinase enzyme. This enzyme facilitates the precise insertion of the GOI into the Flp-In cassette within the cell's genome.

While the second step is straightforward, the initial integration of the Flp-In cassette can be a challenge, relying on the somewhat random nature of DNA integration. To address this, commercially available Flp-In cell lines exist, but they might not always be optimal for expressing specific proteins of interest. This is especially true for proteins needing specific glycosylation patterns, a key feature of Lec 3.2.8.1 CHO cells.

Lec 3.2.8.1 CHO Cells: Developed by Stanley, these cells lack N-type glycosylation due to a mutation in glycosylation enzymes. This results in more homogenous glycosylation of proteins, crucial for applications like protein crystallography.
DG44-CHO Cells: These cells, developed by Chasin and Urlaub, lack the enzyme DHFR, making them dependent on external sources of nucleosides. This allows for selection of cells expressing the DHFR enzyme along with the desired gene, simplifying the process of generating stable cell lines.

The real challenge with Lec 3.2.8.1 and DG44-CHO cells has been achieving stable transfectants with consistent, high-level expression of the desired gene. By creating Flp-In-ready versions of these cells, researchers are making the process significantly easier and more reliable.

Fluorescence-Based Selection: A Game Changer

The key innovation in this research lies in modifying the Flp-In system with a fluorescent marker (mCherryZeo). This allows researchers to use fluorescence-activated cell sorting (FACS) to rapidly identify and isolate cells that have successfully integrated the Flp-In cassette into their genome.

The new pFRT/CherryZeo plasmid is also significantly smaller than its predecessor, making it easier to work with and potentially improving integration efficiency. Using this modified system, the team successfully created Flp-In-ready Lec 3.2.8.1 and DG44 CHO cell lines.

This faster, more reliable method promises to accelerate protein production and open new avenues for research. By streamlining the creation of stable cell lines, researchers can focus on optimizing protein expression and exploring the diverse applications of these valuable cell lines.