Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution

• Published in: Transgenic research Vol. 17; no. 1; pp. 149 - 155
• Main Authors: Sakurai, Takayuki, Kamiyoshi, Akiko, Watanabe, Satoshi, Sato, Masahiro, Shindo, Takayuki
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.02.2008; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Animal Genetics and Genomics; Animals; Base Sequence; beta-Galactosidase - genetics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biotechnology; Crude DNA solution; DNA Primers - genetics; DNA, Recombinant - genetics; DNA, Recombinant - isolation & purification; Female; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Gene-targeted mice; Genetic Engineering; Genetic technics; Heterozygote; Homozygote; Intracellular Signaling Peptides and Proteins - genetics; Life Sciences; Male; Membrane Proteins - genetics; Methods. Procedures. Technologies; Mice; Mice, Knockout; Mice, Transgenic - genetics; Molecular Medicine; Organic Chemicals; Plant Genetics and Genomics; Polymerase Chain Reaction - methods; Pregnancy; Proteins - genetics; Real-time PCR; Receptor Activity-Modifying Protein 2; Receptor Activity-Modifying Protein 3; Receptor Activity-Modifying Proteins; RNA, Untranslated; SYBR Green; Transgenic animals and transgenic plants; Transgenic mice; Transgenics; Zygosity; Crude DNA solution; Gene-targeted mice; Zygosity; Real-time PCR; SYBR Green; Transgenic mice; Targeting; Genomics; Rodentia; Transgenic animal; Real time; Real-time PCR, SYBR Green, Zygosity· Transgenic mice, Gene-targeted mice; Polymerase chain reaction; Vertebrata; Mammalia; Gene; Mouse; DNA
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-007-9134-7

We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses.