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One reason could be that your gene product is toxic to the cells. We recommend testing the construct for expression of the gene of interest by transient Nucleofection™ of your cells. If the proportion of expressing cells drops between 4 and 48 hours...
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Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition.
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The reason is that insect cells are often incubated in very simple incubators that are not humidified (and without CO2 at 24°C to 28°C). Insect media typically do not need CO2 for buffering pH, so gas exchange is not required. To avoid evaporation of...
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This is the normal pattern you should expect to see. Only a small proportion (1/10,000 to 1/100) of all transfected cells will integrate the transfected DNA into their genome and become stable transfectants. The remaining cells lose the transfected...
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The ratio is 1:4.5 (500µL's of Supplement is added to 2.25mL's of Nucleofector solution). For a single reaction, use 18 ul of supplement plus 82 ul of solution to make the 100 ul.
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The most commonly used marker is the neomycin phosphotransferase (neo) gene that confers resistance to G418 to eukaryotic cells. Other markers are Puromycin, Hygromycin, Zeocin, or the HPRT gene that can be used in HPRT-deficient cells.
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Antibiotics should be added 24-48 h after Nucleofection.
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Every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.
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This depends on the respective cell type (transfection efficiency, viability and integration frequency). If it is important to you to know the frequency of stable integrants before carrying out your actual experiments, the frequency should be...
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In vivo, bronchial epithelial cells differentiate along an abnormal squamous pathway under conditions of retinoid deficiency. The squamous phenotype is characterized by the induction of specific markers such as keratin 13, and the enzymes...
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