Abstract
Generation of transgenic mice by DNA microinjection has become one of the most important technologies in studying gene function in vivo. Random integration of transgene often results in insertional mutation which may complicate phenotype analysis of transgenic mice and/or create a good opportunity to study the function of endogenous gene. However, the utilization of these potentially valuable resources is hampered due to lack of efficient approaches for rapid identification of multi-copy transgene integration sites. Here we propose two PCR-based approaches to identifying transgene/genome junction sequences. The efficiency of these two strategies was tested in 9 independent transgenic mouse lines. Among them, the transgene in 8 mouse lines was clearly localized to certain chromosome regions. These rapid and efficient approaches will greatly facilitate the study of insertional mutation due to transgene integration.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Gordon J W, Scangos G A, Plotkin D J, et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Nat Acad USA, 1980, 77(12): 7380–7384
Gordon J W, Ruddle F H. DNA-mediated genetic transformation of mouse embryos and bone marrow—A review. Gene, 1985, 33(2): 121–136
Palmiter R D, Brinster R L, Hammer R E, et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature, 1982, 300(5893): 611–615
Palmiter R D. Germ-line transformation of mice. Ann Rev Genet, 1986, 20: 465–499
Rohan R M, King D, Frels W I. Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes. Nucleic Acids Res, 1990, 18(20): 6089–6095
Pawlik K M, Sun C W, Higgins N P, et al. End joining of genomic DNA and transgene DNA in fertilized mouse eggs. Gene, 1995, 165(2): 173–181
Muller K, Heller H, Doerfler W. Foreign DNA integration. Genome-wide perturbations of methylation and transcription in the recipient genomes. J Biol Chem, 2001, 276(17): 14271–14278
Silver L M. Mouse genetics. New York: Oxford University Press.
Silver J, Keerikatte V. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus. J Virol, 1989, 63(5): 1924–1928
Jena N, Martin-Seisdedos C, McCue P, et al. BMP7 null mutation in mice: Developmental defects in skeleton, kidney, and eye. Exp Cell Res, 1997, 230(1): 28–37
Naora H, Kimura M, Otani H, et al. Transgenic mouse model of hemifacial microsomia: Cloning and characterization of insertional mutation region on chromosome 10. Genomics, 1994, 23(3): 515–519
Zhao X D, Yang W J, Wang L, Kong H et al. Development of salivary gland tumors in pleomorphic adenoma gene 1 transgenic mice. Chin J Med Genet (in Chinese), 2003, 20(5): 390–395
Triglia T, Peterson M G, Kemp D J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of knowing sequences. Nucleic Acids Res, 1988, 16(16): 81–86
Sasaki H, Hamada T, Ueda T, et al. Inherited type of allelic methylation variations in a mouse chromosome region where an integrated transgene shows methylation imprinting. Development, 1991, 111: 573–581
Liu Y G, Whittier R F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics, 1995, 25: 674–681
Hahn S A, Schutte M, Hooue A, et al. A candidate tumor suppressor gene at human 2 chromosome 18q21.1. Science, 1996, 271: 350–353
Rassoulzadegan M, Leopold P, Vailly J, et al. Germ line transmission of autonomous genetic elements in transgenic mouse strains. Cell, 1986, 46: 513–519
Min G S, Powell J R. Long-distance genome walking using the long and accurate polymerase chain reaction. Biotechniques, 1998, 24(3): 398–400
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
Zhao, X., Dang, S., Liang, B. et al. PCR-based approaches for identification of multi-copy transgene integration sites in mouse genome. CHINESE SCI BULL 51, 2231–2235 (2006). https://doi.org/10.1007/s11434-006-2100-6
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11434-006-2100-6