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Indicator and Reporter Dyes
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
Both BAPTA-based small molecule indicators and GECI can be subdivided into two groups: those that provide ratiometric calcium-binding responses and those that do not (Table 8.1). Ratiometric indicators generate fluorescence signals measured at two different excitation (or emission) wavelengths whose ratio is calcium-dependent but is independent of intracellular dye concentration. Ratio measurements therefore provide a means for avoiding false-positive indications of calcium variations that are actually due to unstable or non-uniform intracellular loading, photobleaching and other indicator concentration artifacts.b Generally speaking, the motivation for using ratiometric measurements is more compelling when attempting to compare calcium levels in two or more cells as opposed to measurements where observation is restricted to a single cell. Ratiometric small molecule indicators such as fura-2 require ultraviolet (or two-photon) excitation; longstanding efforts to develop ratiometric calcium indicators with the capacity for excitation at >400 nm have yielded few successes [13]. Ratiometric GECIs are most usually CFP/YFP FRET constructs (Fig. 8.1; [4]). Production of stable cell lines is sometimes impeded by CFP/YFP recombination, resulting in expression of inactive indicators [14].
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Another factor to consider in the selection of an expression vector is antibiotic resistance. Most commercial vectors encode resistance to ampicillin or kanamycin for bacterial selection, and often, vectors are available in both variants. In choosing an antibiotic resistance for expression in E. coli, it is important to consider the E. coli host strain that will be used for expression, since many E. coli strains used for expression already have antibiotic resistance due to genetic manipulation of the host strains. (See below for a discussion.) For expression in mammalian cells, it is often useful to create stable cell lines, which requires the inclusion of a selection cassette within the vector. Common section cassettes for mammalian cell lines are used to select for cells resistant to blasticidin, G418 (Geneticin), Zeocin, and Hygromycin B.
Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells
Published in Preparative Biochemistry and Biotechnology, 2019
Omid Mohammadian, Masoumeh Rajabibazl, Es’hagh Pourmaleki, Hadi Bayat, Roshanak Ahani, Azam Rahimpour
Alternatively, gene transfer strategies based on viral vectors can be employed for the rapid development of stable cell lines.[16] Viral vectors are highly efficient in transferring genetic material into the cells and enabling long-term expression of the transgene. Lentiviruses are RNA viruses from the family Retroviridae. Lentiviral vectors offer several attractive properties including: (i) sustained transgene expression through stable vector integration into the transcription active regions of the host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) the absence of viral proteins expression after vector transduction; (iv) the ability to deliver complex genetic elements; (v) the feasibility of vector manipulation and production; (vi) the ability to transfer up to 8 kb of foreign DNA; and (vii) potentially safer integration site profile compared to retroviral vectors which mainly target active host cell promoters and enhancers.[17–19]