Simple method of zygosity identification in transgenic mice by real-time quantitative PCR.
To determine zygosity in transgenic (Tg) mice, a new technology, real-time quantitative PCR, has recently been introduced in transgenic research to overcome several drawbacks (time-consuming, specialized techniques and/or ambiguity in the results) of previously established methods, for example, Southern blot hybridization, dot blot hybridization, fluorescence in situ hybridization (FISH), etc. However, the previous real-time quantitative PCR method still possesses several drawbacks, for example, it needs two sets of primers/probes and the complicated setting up of appropriate conditions, both of which are expensive and remain time-consuming. We therefore developed an improved real-time quantitative PCR system for determination of zygosity, which is easy, rapid and less expensive, because the technique needs only two experimental processes: estimation of DNA concentration and CYBR Green PCR. We found that homozygous, hemizygous and non-Tg animals could easily be distinguished among F1 littermates in crosses of hemizygous EGFP- and DsRed2-Tg mice. Our improved method will be applicable to any Tg mouse strains, when a primer set is matched to the corresponding transgene.
Shitara
Hiroshi
H
Laboratory for Transgenic Technology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan. shitara@rinshoken.or.jp
Sato
Akitsugu
A
Hayashi
Jun-ichi
J
Mizushima
Noboru
N
Yonekawa
Hiromichi
H
Taya
Choji
C
D000818:Animals; D001483:Base Sequence; D005456:Fluorescent Dyes / Q000032:analysis; D006580:Genetic Carrier Screening / Q000379:methods*; D006579:Heterozygote; D006720:Homozygote; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D005456:Fluorescent Dyes