Abstract
Establishment and maintenance of transgenic mouse strains require being able to distinguish homozygous from heterozygous animals. To date, the developed real-time quantitative PCR techniques are often complicated, time-consuming and expensive. Here, we propose a very easy and rapid method with a simple data analysis to determine zygosity in transgenic mice. We show that the real-time quantitative PCR using SYBR® Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene. Our procedure has to fit only three simple requirements: (1) to design primers capable of detecting one Ct difference for two-fold differences in DNA amounts (2) to measure genomic DNA concentrations accurately and (3) to have a reference animal of known zygosity in each run. Then, if the Ct values for the control gene are similar in all samples, we are able to compare directly the Ct values for the transgene in every sample, and so, to deduce the zygosity status of each mouse relative to the reference animal. This method is really simple and reliable, and it may be valuable as a rapid screening tool for zygosity status in transgenic animals.
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References
Bubner B, Baldwin IT (2004) Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep 23:263–271
Bubner B, Gase K, Baldwin IT (2004) Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR. BMC Biotechnol 4:14
Ji W, Zhou W, Abruzzese R, Guo W, Blake A, Davis S, Polejaeva I (2005) A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR. Anal Biochem 344:240–246
Prior FA, Tackaberry ES, Aubin RA, Casley WL (2006) Accurate determination of zygosity in transgenic rice by real-time PCR does not require standard curves or efficiency correction. Transgenic Res 15:261–265
Shitara H, Sato A, Hayashi J, Mizushima N, Yonekawa H, Taya C (2004) Simple method of zygosity identification in transgenic mice by real-time quantitative PCR. Transgenic Res 13:191–194
Tesson L, Heslan JM, Menoret S, Anegon I (2002) Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Res 11:43–48
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We would like to thank M. Allard and C. Chevalier for technical assistance.
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Haurogné, K., Bach, JM. & Lieubeau, B. Easy and rapid method of zygosity determination in transgenic mice by SYBR® Green real-time quantitative PCR with a simple data analysis. Transgenic Res 16, 127–131 (2007). https://doi.org/10.1007/s11248-006-9024-4
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DOI: https://doi.org/10.1007/s11248-006-9024-4