Geneticin, G-418 Sulfate

Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing

With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.

Efficient selection for high-expression transfectants with a novel eukaryotic vector

We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.

Geneticin reduces mRNA stability

Messenger RNA (mRNA) translation can lead to higher rates of mRNA decay, suggesting the ribosome plays a role in mRNA destruction. Furthermore, mRNA features, such as codon identities, which are directly probed by the ribosome, correlate with mRNA decay rates. Many amino acids are encoded by synonymous codons, some of which are decoded by more abundant tRNAs leading to more optimal translation and increased mRNA stability. Variable translation rates for synonymous codons can lead to ribosomal collisions as ribosomes transit regions with suboptimal codons, and ribosomal collisions can promote mRNA decay. In addition to different translation rates, the presence of certain codons can also lead to higher or lower rates of amino acid misincorporation which could potentially lead to protein misfolding if a substituted amino acid fails to make critical contacts in a structure. Here, we test whether Geneticin-G418, an aminoglycoside antibiotic known to promote amino acid misincorporation-affects mRNA stability. We observe that G418 decreases firefly luciferase mRNA stability in an in vitro translation system and also reduces mRNA stability in mouse embryonic stem cells (mESCs). G418-sensitive mRNAs are enriched for certain optimal codons that contain G or C in the wobble position, arguing that G418 blunts the stabilizing effects of codon optimality.

Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells

Conventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are not tumor site-specific and have cytotoxic and harmful side effects for normal cells. Mesenchymal stem cells (MSCs), due to their tumor-tropism migration property, are a promising alternative to deliver and produce antitumor agents. However, MSCs are difficult-to-transfect cells, and introducing the exogenous therapeutic gene into MSCs is challenging yet needs improvement. Transfection using chemical reagents, including Lipofectamine, is more convenient and less cytotoxic compared with different methods of introducing exogenous DNA into MSCs. Nonetheless, the major limitation of Lipofectamine is low transfection efficiency in MSCs. Therefore, the purpose of this study was to evaluate and suggest the optimum quantities of lipoplex components to enhance the transfection efficiency of human adipose tissue-derived MSCs (hASCs). Finding the best transgene expression time point and the optimum concentration of G-418 for antibiotic-based selection was another goal of this study. hASCs were transfected in a series of experiments with altering the quantities of Lipofectamine LTX? (Lip-LTX), the related "PLUS" reagent, and a plasmid DNA (pDNA) expressing the enhanced green fluorescent protein (eGFP). After transfection, the percentage of eGFP-expressing cells was evaluated using fluorescence microscopy and ImageJ software in 12-hour intervals for 48 hours. Also, the viability of hASCs exposed to different concentrations of G-418 was measured using an MTT assay. The results demonstrated that a combination of 2 ?L Lip-LTX, 0.75 ?L of its "PLUS" reagent, and 0.75 g pDNA (6484 bp) improve the transfection efficiency of hASCs (23.75%), and the best period for evaluation of fluorescence for these cells is 12 to 24h post-transfection. Also, the optimum concentration of G-418 for antibiotic-based selection of hASCs was 0.25mg/mL. In conclusion, this study indicates that the setting up of optimized quantities of lipoplex components and the golden time of evaluation for transgene expression could increase the possibility of transgene expression in hASCs before beginning research and clinical application. Also, the definition of optimal dose of selection antibiotic for purification of transfected hASCs seems to be necessary for maximum transgene expression effects in the cell population.

A breakthrough in readthrough? Could geneticin lead the way to effective treatment for cystinosis nonsense mutations?