Site-Directed Engineering of Defined Chromosomal Sites for Recombinant Protein and Virus Expression - Site-directed engineering of defined chromosomal sites. - BioPharm International


Site-Directed Engineering of Defined Chromosomal Sites for Recombinant Protein and Virus Expression
Site-directed engineering of defined chromosomal sites.

BioPharm International
Volume 22, Issue 7


We screened various cell lines including human 293 cells, mouse NIH3T3, Chinese hamster ovary (CHO), and baby hamster kidney (BHK) cells for integration sites that meet the requirements of stable and high expression. For this purpose, we implemented an advanced site-specific cassette replacement strategy that combines retroviral tagging and a positive selection trap with the Flp/FRT recombination.9,10 To select exclusively cells that underwent the site-specific recombination, the tagging cassette contains a transcriptionally inactive selection marker that is only activated by correct site-specific integration. This strategy makes it possible to correctly identify recombined clones with an efficiency level of 90% or better.10 We established a set of cells with singly tagged chromosomal sites and used green fluorescent protein (GFP) to identify potent integration sites. Various tagging protocols were successfully applied.11

Figure 3A. Predictable and homogeneous product expression after targeting a tagged locus A. The GFP-containing tagging cassette of a master cell line has been replaced by a targeting vector encoding lacZ. The GFP expression analysis (measured by FACS) and a lacZ staining protocol were performed before and after RMCE. Whereas the tagged cell population exhibits a high level GFP expression but is negative in lacZ staining, resulting subclones after RMCE homogeneously lost the GFP signal but gained the expression of lacZ.
After the sites are tagged and characterized, they can be used to target expression cassettes of choice. Figure 3A gives examples for a GFP-tagged cell line into which the lacZ expression cassette was integrated. Targeting eliminates the GFP and includes lacZ. Importantly, the clones generated upon targeting have homogeneous expression profiles as expected from the integration into a given chromosomal site.

Figure 3B. Predictable and homogeneous product expression after targeting a tagged locus B. Seven individual clones of the RMCE experiment described in A were analyzed by Southern blotting to confirm a correct cassette exchange. Therefore, chromosomal DNA of the clones was digested with DNA endonucleases, blotted, and probed against the marker gene. Only successfully targeted clones would show a 3 kb fragment and be clearly distinguished from the tagged parental clone The lacZ expression of these clones was quantified and compared to the parental cell line. As shown in the diagram, the expression behavior is highly homogenous among all analyzed isogenic subclones.
We exploited this homogenous expression to generate retroviral producer cells. The production of viruses, or viral vectors, can be seen as a special case of protein expression. Retroviral vectors constitute a powerful tool for stable gene transfer into mammalian cells. They can be used to efficiently infect cells of different origin of various species. Vectors derived from murine leukemia virus (MLV) are used as gene delivery systems in clinical gene therapy trials.12 Considerable success in the treatment of several inherited diseases by integrating an expression unit for the therapeutic transgene(s) in the cellular genome delivered by retroviral vectors has been achieved, demonstrating the enormous potential of viral vectors in clinical therapy.13–15

To translate the application of viral vectors into the clinic, it is mandatory to produce high titers of viral vector from a fully characterized producer cell line. For each therapeutic vector, a specific producer cell line must be generated. The titer critically depends on the site of chromosomal integration of the therapeutic vector. Thus, tremendous screening efforts must be undertaken to isolate clones that express the vectors in high concentrations and stably over time—a process that can take several months when conventional retrovirus packaging cell lines are used.16 We established a strategy based on RMCE to establish a new generation of producer cell lines. Upon selection for a chromosomal locus that supports retroviral vector insertion, a flexible retrovirus producer cell line was established that simplifies the isolation of highly productive producer clones.3,4 This approach reduces laboratory time, increases safety, and in particular makes it possible to adjust viral vectors to the requirements of individual loci.17

blog comments powered by Disqus



Bristol-Myers Squibb and Five Prime Therapeutics Collaborate on Development of Immunomodulator
November 26, 2014
Merck Enters into Licensing Agreement with NewLink for Investigational Ebola Vaccine
November 25, 2014
FDA Extends Review of Novartis' Investigational Compound for Multiple Myeloma
November 25, 2014
AstraZeneca Expands Biologics Manufacturing in Maryland
November 25, 2014
GSK Leads Big Pharma in Making Its Medicines Accessible
November 24, 2014
Author Guidelines
Source: BioPharm International,
Click here