Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome.

Kazuya Sakata,Hiroyasu Nakano,Kimi Araki,Anna S Gukovskaya,Sachiko Komazawa-Sakon,Takashi Nishina,Ken-Ichi Yamamura,Grace E Lee,Masaki Ohmuraya,Daisuke Hashimoto,Chigure Suzuki,Ilya Gukovsky,Hideo Baba,Kenji Notohara,Yasuo Uchiyama,Shin Murai

doi:10.1038/srep37200

Abstract

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed “X-SPINK1“. Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/− mice rescued perinatal lethality, but the resulting Spink3−/−;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3−/−;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.

Highlights

These cells do not display chromatin condensation, a hallmark of apoptosis)[3]
Taken into account that Diaphanous homolog 2 (Diap2)-deficient mice develop without abnormalities and are fertile[11], we reckoned that integration and expression of a target gene in the Diap[2] locus on X chromosome might be feasible by using Cre-loxP technology; and further, that this approach would allow us to express the target gene in a mosaic pattern due to random inactivation of one of the two X chromosomes in females
We have developed a novel method to express a target gene in a mosaic pattern through its’ specific integration onto the X-chromosome by using Cre-loxP technology

Summary

Introduction

These cells do not display chromatin condensation, a hallmark of apoptosis)[3]. The early death of Spink3−/− mice precludes investigation into the mechanisms of long-term effects of SPINK1 deficiency. Once the X chromosome is inactivated, it will remain inactive throughout the lifetime of the cell. During screening of a gene trap library, we found that one ES cell line (B210) possessed a trap vector on the X chromosome. By utilizing X-chromosome inactivation and the B210 ES cells, we here present a novel method to efficiently integrate a target gene on the X chromosome, resulting in a mosaic pattern of the target protein expression. This strategy enabled us to rescue perinatal lethality of Spink3−/− mice. The resulting Spink3−/−;XX SPINK1 mice developed spontaneous pancreatitis, representing a novel genetic model for this disease

Methods

Results

Conclusion