Characterization of a bicistronic knock-in reporter mouse model for investigating the role of Cables2 in vivo

概要

Introduction and Purpose
The CDK5 and Abl enzyme substrate (Cables) family comprises two members, Cables1 and Cables2. Both proteins have a cyclin box-like domain at the C-terminus, which is highly homologous as it presents 78% amino acid identity. Cables family members interact physically and associate with cyclin-dependent kinase 3 (Cdk3), Cdk5, and c-Abl. Cables1 plays a key regulatory role in human intestinal tumor progression, endometrial hyperplasia, and oocyte development. Furthermore, Cables1 is a mediator for progesterone-induced differentiation of endometrial epithelial cells. CABLES1 is up- regulated by progesterone, but down-regulated by estrogen. Further, Cables1 is associated with protecting p63 from protein degradation and maintaining p21/Cip1 stability. In addition, Cables1 induces p53- and p73-mediated apoptosis, while binding to 14-3-3 supresses the apoptosis-inducing function of Cables1. In vitro, Cables2 induces apoptotic cell death in both a p53-dependent and a p53-independent manner. Cables2 mRNA is found to be widely expressed in adult mouse tissues by northern blot analysis. Although Cables family members share a similar protein structure, the role of Cables2 in vivo remains unknown, largely due to a lack of suitable antibodies against mouse Cables2 and absence of a Cables2 mouse model.

In this present study, I created and characterized a novel knock-in mouse that can boost our understanding of Cables2 expressing cells, protein interaction network, and functions in vivo. I generated bicistronic Cables2 knock-in reporter mice that expressed Cables2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Hereafter, Cables2-3×FLAG-2A-tdTomato mouse is referred to as Cables2Tom.

Materials and Methods
Cables2Tom mouse was generated by modifying Cables2 gene in embryonic stem cells (ES cells) using the CRISPR/Cas9 system. I knocked-in 3xflag, 2A, and tdTomato just before stop codon of Cables2. To investigated whether the Cables2Tom mouse can provide a valuable tool in studying Cables2, I subjected it to several different analyses. Expression of Cables2 RNA was determined by RT-PCR and RT-qPCR. Visualizing of Cables2 in mouse organs and tissues was examined by observing tdTomato fluorescent signal in fixed samples. Expression of Cables2 protein was evaluate by Western blotting. Cables2 protein-protein interactions with Cdk5 in Cables2Tom mice was evaluated by IP. Furthermore, serum progesterone concentrations in pregnant Cables2Tom mice were measured by ELISA.

Results and Discussion
First, I confirmed targeted gene insertion and homologous recombination of ES cells clones and I evaluated random integration in these clones. Then I characterized Cables2Tom mice by subjecting the knock-in mice to several analyses. RT-PCR analysis detected tdTomato band in the brain, lung, kidney, spleen, colon, testis, and ovary from Cables2Tom mice but not from wild-type mice. RT-qPCR analysis showed significant increase of Cables2 expression level in the brain and testis from Cables2Tom mice comparing to wild-type mice. Fluorescent signal was higher in the brain, testis and ovary from Cables2Tom mice comparing to wild-type mice. Interestingly, unique expression pattern was observed in corpus luteum of the ovary. By using anti-FLAG antibody, western blot showed flag band in the brain, testis and ovary from Cables2Tom mice but not wild-type mice. Immunoprecipitation analysis using the brain and testis in Cables2Tom revealed interaction of Cables2 with Cdk5. These results suggest that Cables2Tom mouse is a useful tool to study Cables2 in vivo.

Finally, to progress in understanding the functional roles of Cables2 in the corpus luteum, I investigated whether Cables2 is one of the molecules involved in the luteinization process by using Cables2Tom mice. The corpus luteum is an endocrine structure that secretes progesterone hormone which prepares the endometrium for possible implantation. I confirmed whether Cables2Tom mouse is an effective overexpression model for investigating Cables2 in the ovary. At gestation day 15.5, RT- qPCR analysis revealed that Cables2 mRNA expression in the ovary was increased at approximately 4 times in Cables2Tom mice compared with wild-type mice. Strong tdTomato signals were observed in the corpus luteum of pregnancy. Measuring the number of viable fetuses Cables2Tom mice obtained by inbreeding was significantly less than that in wild-type. Further, Cables2Tom mice showed significantly lesser ovary weight than wild-type mice at gestation day 15.5. Surprisingly, although pregnant Cables2Tom mice that overexpress Cables2 mRNA in organs including the ovary showed less in litter size and ovary weight than pregnant wild-type mice, serum progesterone concentrations were significantly higher in pregnant Cables2Tom mice. These results imply that Cables2 is a functional molecule involved in progesterone biosynthesis process.

Conclusion
Collectively, I demonstrate that our bicistronic Cables2 knock-in reporter mouse is a useful model for the comprehensive analysis of in vivo Cables2 function, particularly in the corpus luteum.