Creation of Custom-Engineered Cell Lines by Our Cell Design Studio™ Provides Key Tools for Researchers | Biowire Spring 2012

By: Gregory Wemhoff, Principal Scientist; Erika Holroyd, Scientist; Bradley Keller, Product Manager, Biowire Spring 2012, 6–9

Biowire Spring 2012 — Live Cell Imaging of Signaling Pathways

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The onset of cancer is generally believed to occur from the accumulation of specific mutations that alter the genetic makeup of the transformed cell1. Some mutations may result in over- or under-expression of the target gene, while other mutations may be as subtle as a single base pair change altering ligand/substrate interaction. Early studies demonstrated the loss of TP53 gene expression leads to a significantly higher tumorigenic rate compared to cells with normal expression2. Moreover, since TP53 was first recognized as a tumor suppressor, the number of genes identified as playing a role in cancer development has increased dramatically. Recently, the advent of robust, high-throughput, and cost-efficient methods of sequencing DNA has accelerated the rate of identification of specific cancer-relevant mutations and the use of transformed cell lines to study these mutations.

The hypotriploid, transformed human lung epithelial line, A549, has been used extensively to study lung cancer and carries mutations in several genes known to be associated with cancer development3 such as the epithelial growth factor receptor (EGFR). In the review of Sharma et al.4, over 40 different mutations associated with EGFR were highlighted. The precise EGFR mutation a patient carries is critical, rendering the tumor more or less susceptible to kinase inhibitors such as gefitinib and erlotinib5, and ultimately affecting the choice of treatment. Unfortunately, panels of cell lines that allow investigation of each EGFR mutation or other cancer-relevant mutations are not currently accessible. Using zinc finger nuclease (ZFN) technology, cell lines containing very specific, directed mutations can be created. The basic researcher, as well as diagnostic and therapeutic development programs, need no longer be saddled by the limited availability of relevant cell lines for their studies. Through Sigma Life Science’s Cell Design Studio™ (CDS) custom cell line development group, cell lines containing mutations of interest can be generated.

To initiate an inquiry, a researcher approaches CDS and requests a specific cell line carrying a targeted gene with their modification of interest (Figure 1). CDS then investigates the target cell line to determine clonability (i.e., expansion from a single-cell parent), chromosome ploidy of the target gene, potential target gene amplification, and other key features. Modifications of the target gene can include base insertions, deletions, or nucleotide base substitutions to mimic naturally occurring mutations. Fusion proteins can also be created for the purpose of attaching reporter tags to an endogenous gene locus. Notably, genetic information can be introduced into welldefined locations, rather than relying on random integration events, thus reducing the impact on expression of endogenous genes. Control is paramount, as the genetic alterations can occur on a single allele, leaving the other allele(s) in the wild-type state (i.e., a heterozygous mutation), or all alleles can be altered (i.e., homozygous). Unlike limitations associated with other gene engineering technologies, ZFN -based alterations can alter every allele in polyploid cell lines.

Workflow for a Standard CDS Cell Line Project

Figure 1. Workflow for a Standard CDS Cell Line Project.

As an example of the CDS process, let’s assume an investigator involved with breast cancer research requires a breast epithelial cell line that carries a particular mutation known as the BRAF mutation. It is well documented that in melanoma and colorectal carcinoma the BRAF gene often carries a single nucleotide change, the V600E mutation, which is thought to promote metastasis. Using a nearnormal breast epithelial line, MCF10A, the CDS team first pursues the generation of a ZFN pair that will cut specifically near the site where the V600E mutation is known to occur. Additionally, a donor oligonucleotide sequence is designed having the single nucleotide difference associated with the V600E mutation and flanked by a wild-type (homologous) sequence. Concurrently, investigations are conducted to examine ploidy of the MCF10A cell line, which reveal it is diploid, thus having only two alleles for the BRAF gene target. If not previously examined by the CDS, the cell line is subjected to single-cell cloning to determine the difficulty of generating clonal cell populations. In the case that the cells cannot be grown from singlecell clones, the customer is advised, and it is determined whether a mixed population will serve their needs. The target gene is also investigated (through available literature) regarding the potential impact on cell viability if it is disrupted. This includes understanding if the desired mutation is considered a heterozygous dominant mutation such that, if only a single allele carries the mutation, the oncogenic effect is realized. With the key information in hand, the customer is contacted to determine if they require a homozygous or a heterozygous alteration of their gene target, after which work to create the cell line may commence.

Once available, the ZFNs with the “donor oligonucleotide” carrying the BRAF mutation are nucleofected into the MCF10A cell line, and ZFN activity is confirmed from samples of the cell pool. The cells are then expanded and subjected to single-cell sorting to generate cloned cell lines. Each clonal line is expanded and subsequently screened by sequence analysis to determine which are carrying the desired mutation. The clones screening positive for the mutation are examined further to determine if all alleles carry the mutation or if a fraction of the alleles have been impacted. Specific populations of the clones are then selected for further expansion and cryopreservation.

At numerous steps throughout the CDS process, the customer is advised of the experimental results and must determine if the project should continue to move forward. One possible outcome, after attempting two nucleofections, is the inability to isolate cells carrying the desired mutation due to the complexity of the biology (e.g., gene polyploidy). A customer must then decide if additional nucleofections will be attempted or if they would prefer terminating the current project and attempting a different approach. Varying approaches could include selecting a different ZFN pair targeting a slightly different cut site, designing a different donor oligonucleotide, or perhaps pursuing a different target cell line.

While this example outlines the introduction of a single nucleotide mutation, the insertion of stop codons to shut down gene translation or the generation of out-of-frame insertions or deletions may also be pursued to alter gene expression. Additionally, over-expression of genes or gene amplification can be mimicked by introducing an additional copy in “safe harbor” sites known to have no impact on cell integrity, such as the AAVS1 site6. Table 1 illustrates several types of projects the CDS team can conduct for customers.

General Types of Projects Conducted by the CDS

Table 1. General Types of Projects Conducted by the CDS.

The ability to use either normal or near-normal cell lines instead of transformed lines allows the researcher to investigate a particular gene target against a near-normal background. This is quite distinct from using a transformed parental cell line where several genes are known to be disrupted and, in many instances, present in a polyploid state. To that end, our CDS expertise is also available for modification of inducible pluripotent stem (iPS) cells to create a “fit-for-purpose” cell line. In addition to the full range of gene-editing capabilities, characterization of iPS cells such as directed differentiation, copy number variation analysis, and epigenetic analysis can be performed. Just as with the immortalized cell lines, deliverables include the cell line, the ZFN kit (and donor if applicable), and complete project report documentation to support scientific publications.

This article began by outlining cancer generally as the result of “the accumulation” of various gene-altering events. The CDS team has the ability to introduce multiple, distinct modifications into the target cell line. Following the example, once a clone carrying the BRAFV600E mutation has been isolated, this cell line can then serve as the foundation line to introduce additional mutations. Many cancers carry a loss of PTEN expression in addition to other mutations, and colorectal cancers are often V600E-positive and carry a KRAS mutation. An additional option available through the CDS group is to tag genes with markers such as fluorescent proteins. Cells can then be readily tracked and the migration of cellular proteins monitored through live cell imaging, a method ideal for high-content analysis screening. While the target cell line or target gene may introduce some limitations, the overall possibilities for cell line creation by the CDS are limited primarily by the expressed desire of the researcher. The next step to advancing a treatment or cure for a cancer, or other genetic condition, may first be identified by your desired gene modification in a new cellular disease model developed by the CDS.

 

 References

  1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144(5):646–74.
  2. Lane DP, Crawford LV . T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979;278(5701):261–63.
  3. Chang H, Jackson DG, Kayne PS, et al. Exome sequencing reveals comprehensive genomic alterations across eight cancer cell lines. PLoS One. 2011;6(6):e21097.
  4. Sharma SV, Bell DW, Settleman J, et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7(3):169–81.
  5. Janne PA. Challenges of detecting EGFR T790M in gefitinib/erlotinib-resistant tumors. Lung Cancer. 2008;Suppl2:S3–9.
  6. Lombardo A, et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods. 2011;8(10):861–9.

 

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