Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes |
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| Authors: | Cecilia Mancini Erika Messana Emilia Turco Alessandro Brussino Alfredo Brusco |
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| Affiliation: | (1) Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy;(2) S.C.D.U. Medical Genetics, A.O.U. San Giovanni Battista, Torino, Italy;(3) Molecular Biotechnology Center, Torino, Italy |
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| Abstract: | In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene. |
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