Int. J. Dev. Biol. 49: 807 - 823 (2005)

https://doi.org/10.1387/ijdb.051975mi

Vol 49, Issue 7

Gene disruption/knock-in analysis of mONT3: vector construction by employing both in vivo and in vitro recombinations

Open Access | Original Article | Published: 1 September 2005

Makoto Ikeya^1,*, Masako Kawada¹, Yoko Nakazawa¹, Makoto Sakuragi¹, Noriaki Sasai¹, Morio Ueno¹, Hiroshi Kiyonari², Kazuki Nakao² and Yoshiki Sasai¹

¹Organogenesis and Neurogenesis Group and ²Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe, Japan

Abstract

We report the isolation, spatial/temporal expression and gene disruption phenotype of the mouse ONT3 (mONT3) gene, which encodes a novel secreted signaling protein belonging to the Olfactomedin/Noelin/Tiarin family. During early embryogenesis, mONT3 is detected in the proximal region of the allantois on embryonic day (E) 7.25, in the lateral plate mesoderm on E 8.0 and in the CNS and heart on E 8.5. The homozygous mutant is born normal and fertile. For the expression pattern and loss-of-function analyses, we have successfully generated the LacZ-knock-in targeting vector directly from BACs carrying mouse genomic fragments by combining in vivo and in vitro recombination techniques. This approach enables rapid and reproducible construction of the fully functional vectors within two weeks without the use of restriction enzyme digestion and ligation, or the use of PCR-amplification of large genomic fragments. In addition, this method is applicable to rapid generation of transgenic vectors, demonstrating its versatility in reverse genetic studies.

Keywords

ONT, EG construction, gene expression, knock-in, recombination