The overall goal of this procedure is to use a custom engineered zinc finger nuclease or ZFN to introduce a disruptive mutation at a specific locus to ultimately create a knockout cell line. This is accomplished by first analyzing the DNA sequence of the target gene with the composer algorithm to generate insco ZFN designs. The Z fns are assembled delivered to cells by NU nuclear affection and screened for activity at their respective gene target sites for generating a knockout cell line.
The cell type of interest is first treated with an active ZFN. Then this population is segregated to single cells. And finally, these clonal populations are expanded for additional genotypic characterization Clones with disruptive non-homologous and joining events at the target site may be studied further in the context of functional gene knockout.
Ultimately, genetic characterization, including the cell one assay and DNA sequencing are necessary to confirm, observed changes in cellular phenotype as a result of ZFN mediated genetic modification. The main advantage was zinc finger nucleuses relative to existing methods like traditional homologous recombination is composer ZFN enabled. Gene editing allows researchers to create specific and predictable genetic modifications without selection.
This includes gene knockouts, gene modifications, and gene integrations. The ZFN design process begins with simplified bioinformatic studies, which focus on identifying unique regions of sequence containing sufficient number and quality of zinc finger binding motifs that are conducive to performing gene knockout. A clearly stated experimental strategy will greatly facilitate this process, as will information on the target gene, such as atypical gene structure, special biology implicated in the genetic locus, and any homologous regions elsewhere in the genome.
For more complex projects, a sigma scientist will work closely with the investigators. The Z fns are designed in silico and the whole genome off-target sites are predicted. Repeat masking and snip analysis are also performed for each potential ZFN target site.
The top ZFN target sites are reviewed before ZFN production. Researchers often ask, will ZF N'S work for my project? To best address this question, we should take into account several key considerations.
Can you deliver efficiently and express a typical expression cassette in your cell type of interest? Can you isolate and subsequently expand single cell clones? And finally, is there a deleterious impact on the cell growth or cell viability as a result of knocking out your gene of interest?
Answers to these questions may or may not strictly dictate a project's feasibility. However, they allow us to gauge how easy or how hard a particular project might be, or they may shed light on particular aspects of the workflow that are critical to success. Sigma uses its archive of zinc finger modules to assemble the approved ZFN designs.
Each ZFN design is assembled and sequence verified on a high throughput cloning platform. Then ZF NS are delivered in plasmid DNA format to the relevant well-characterized cell line and become sufficiently expressed in 24 to 48 hours. Genomic DNA is then harvested from each pool of ZFN treated cells, followed by PCR amplification of the ZFN target site.
A cell one assay is conducted on the PCR products and ZFN activities assessed by gel electrophoresis. The highest activity ZFN design is delivered. Additionally, positive control genomic DNA is provided along with PCR primers for reproducing the results shown in the certificate of analysis.
To start your ZFN experiment, begin by seeding the cells at a density of 200, 000 cells per milliliter the day before their nu nuclear affection. The next day count the cells proceed. If their density is between 250 and 500, 000 cells per milliliter, prepare the NU nuclear affection solution V for the cell line you're working with according to the manufacturer's protocol.
Now, fill a six well plate with two milliliters of medium per well and warm the plate in an incubator for at least 20 minutes while warming the plate Centrifuge 2 million cells per nucle affection. Remove the supra named and gently resuspend the cells in 10 milliliters of HBSS. Then repeat the wash procedure once or twice more, ZF NS delivered an mRNA format, typically yield higher gene editing frequencies than those same ZF NS delivered in plasma DNA format.
However, delivering ZF NS in mRNA format requires us to take a few extra precautions to avoid degradation. First, wash your cells with two to three large volumes of PBS or HBSS to remove serum derived RNAs. Also, always keep your mRNA on ice during the entire course of the experiment.
Last, be sure to add the mRNA to your cells at the very last possible moment to avoid degradation. Next, prepare the experimental tubes according to the table in the custom ZFN tech bulletin. For each transfection, re suspend the cells in 100 microliters of nuclear affection solution V.Preparing one reaction at a time.
Add 100 microliters of cells to each DNA or mRNA containing tube. Then transfer each mixture to a two millimeter electroporation. Qve and nucle affect the cells with the appropriate program immediately after the nucle affection of each sample.
Use a transfer pipette to add one half milliliter of warmed medium from a well of the prepared six wall plate. Carefully transfer the cells from the qve back to the remaining medium. In that well finish all reactions and return the plate to the incubator, one to three days.
Post nucleo affection isolate genomic DNA using the genal loop mammalian genomic DNA mini prep kit without harvesting all the pooled cells. Next, use the supplied primers to PCR. Amplify the genomic DNA from the ZFN Transfected samples and the positive control genomic DNA provided in the kit.
Digest each PCR product with one microliter of enhancer and one microliter of nuclease s. Incubate them at 42 degrees Celsius for 20 to 40 minutes. Then run the digestions on a 10%page TBE gel with proper markers such as direct load wide range DNA ladder after single cell cloning the ZFN treated samples and allowing the clones to expand sufficiently.
Harvest a portion of the cells for genomic DNA, continue culture of the remaining cells or freeze back as banked material. Amplify the genomic DNA using the cell one primers and analyze the product by cell one assay with and without wild type amplicon added at a one-to-one ratio. Alternatively, use this amplicon to proceed directly to genotyping genotype candidate clones by sequencing or other preferred methods.
In this example, the cell one assay was used to estimate the activity of a pair of ZF NS and K 5 62 cells. The glan represents the positive control and the Z lane shows cut products from which the frequency of mismatched PCR fragments is estimated from these data and estimation of ZF N activity came to 21%which is very high. Here we show the activity of a different pair of Z fns.
This ZFN pair was significantly less active than the other, but still effective. It is estimated to have created indels in 2.4%of the cell population. Some of the more critical parameters around working with ZF X include optimization of delivery and expression in your cell type.
By optimizing these parameters, one can substantially decrease the amount of time and effort required to isolate the gene knockout. Clone Z fns represent a very precise tool for targeted genome editing used correctly and in the appropriate context. They can provide scientists with a very fast and deficient means for studying the biology in most any gene product or pathway.
