Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay
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2014
Članak u časopisu (Objavljena verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt
The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target: control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.
Ključne reči:
Transgene copy number / PCR / Fluorescently labeled primers / Capillary electrophoresisIzvor:
Biochemical Genetics, 2014, 52, 3-4, 159-165Izdavač:
- Springer/Plenum Publishers, New York
Finansiranje / projekti:
- Kompleksne bolesti kao model sistem za proučavanje modulacije fenotipa-strukturna i funkcionalna analiza molekularnih biomarkera (RS-MESTD-Basic Research (BR or ON)-173008)
DOI: 10.1007/s10528-013-9636-3
ISSN: 0006-2928
PubMed: 24292648
WoS: 000332800800004
Scopus: 2-s2.0-84898824528
Institucija/grupa
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Pruner, Iva AU - Đorđević, Valentina AU - Gvozdenov, Maja AU - Tomić, Branko AU - Radojković, Dragica PY - 2014 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/787 AB - The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target: control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments. PB - Springer/Plenum Publishers, New York T2 - Biochemical Genetics T1 - Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay EP - 165 IS - 3-4 SP - 159 VL - 52 DO - 10.1007/s10528-013-9636-3 ER -
@article{ author = "Pruner, Iva and Đorđević, Valentina and Gvozdenov, Maja and Tomić, Branko and Radojković, Dragica", year = "2014", abstract = "The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target: control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.", publisher = "Springer/Plenum Publishers, New York", journal = "Biochemical Genetics", title = "Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay", pages = "165-159", number = "3-4", volume = "52", doi = "10.1007/s10528-013-9636-3" }
Pruner, I., Đorđević, V., Gvozdenov, M., Tomić, B.,& Radojković, D.. (2014). Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay. in Biochemical Genetics Springer/Plenum Publishers, New York., 52(3-4), 159-165. https://doi.org/10.1007/s10528-013-9636-3
Pruner I, Đorđević V, Gvozdenov M, Tomić B, Radojković D. Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay. in Biochemical Genetics. 2014;52(3-4):159-165. doi:10.1007/s10528-013-9636-3 .
Pruner, Iva, Đorđević, Valentina, Gvozdenov, Maja, Tomić, Branko, Radojković, Dragica, "Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay" in Biochemical Genetics, 52, no. 3-4 (2014):159-165, https://doi.org/10.1007/s10528-013-9636-3 . .