Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR

被引：1

作者：

Fu, P ^{[1
]}

Senior, P ^{[1
]}

Fernley, RT ^{[1
]}

Tregear, GW ^{[1
]}

Aldred, GP ^{[1
]}

机构：

[1] Univ Melbourne, Howard Florey Inst Expt Physiol & Med, Parkville, Vic 3052, Australia

来源：

JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS | 1999年 / 40卷 / 03期

基金：

英国医学研究理事会;

关键词：

transgene copy number; competitive PCR; parathyroid hormone-related protein (PTHrP); murine erythroleukemia (MEL) cells;

D O I：

10.1016/S0165-022X(99)00018-4

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10(4) cells are required for the analysis. (C) 1999 Elsevier Science B.V. All rights reserved.

引用

页码：101 / 112

页数：12

共 18 条

[11]

MOLESH DA, 1994, PCR METH APPL, V3, P278

[12] LCR/MEL - A VERSATILE SYSTEM FOR HIGH-LEVEL EXPRESSION OF HETEROLOGOUS PROTEINS IN ERYTHROID-CELLS [J].

NEEDHAM, M ;

GOODING, C ;

HUDSON, K ;

ANTONIOU, M ;

GROSVELD, F ;

HOLLIS, M .

NUCLEIC ACIDS RESEARCH, 1992, 20 (05) :997-1003

[13]

Ruano G, 1992, PCR Methods Appl, V2, P112

[14]

Sambrook J., 2002, MOL CLONING LAB MANU

[15] A DOMINANT CONTROL REGION FROM THE HUMAN BETA-GLOBIN LOCUS CONFERRING INTEGRATION SITE-INDEPENDENT GENE-EXPRESSION [J].

TALBOT, D ;

COLLIS, P ;

ANTONIOU, M ;

VIDAL, M ;

GROSVELD, F ;

GREAVES, DR .

NATURE, 1989, 338 (6213) :352-355

[16]

Uberla K, 1991, PCR Methods Appl, V1, P136

[17] QUANTITATION OF MESSENGER-RNA BY THE POLYMERASE CHAIN-REACTION [J].

WANG, AM ;

DOYLE, MV ;

MARK, DF .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (24) :9717-9721

[18] RAT PARATHYROID HORMONE-LIKE PEPTIDE - COMPARISON WITH THE HUMAN HOMOLOG AND EXPRESSION IN MALIGNANT AND NORMAL TISSUE [J].

YASUDA, T ;

BANVILLE, D ;

RABBANI, SA ;

HENDY, GN ;

GOLTZMAN, D .

MOLECULAR ENDOCRINOLOGY, 1989, 3 (03) :518-525

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