The promoter region of 46-kDa CNPase is sufficient for its expression in corpus callosum.

Myelin is formed by oligodendrocytes in the central nervous system (CNS) or by Schwann cells in the peripheral nervous system (PNS). It is composed of many bioorganic components such as lipids, proteins and amino acids, as well as nucleotides [1,2]. Growing evidence indicates that myelin diseases represented by genetic hypomyelinating leukodystrophies (HLDs) are related to the failure of the metabolism [3,4].

2’,3’-Cyclic nucleotide phosphodiesterase (CNPase) is one of the major myelin component proteins in the CNS. CNPase participates not only in nucleotide metabolism as intracellular phosphodiesterase but also in linking actin cytoskeletons to the intracellular side of myelin membranes [1]. The cnpase gene encodes two isoforms of 48- and 46-kDa, which are regulated by different promoters [5]. Despite the important role of CNPase in myelin membrane homeostasis, the question of whether the isolated 1-kilobase upstream unit from mRNA encoding the smaller isoform actually contributes to protein expression remains to be unanswered. In contrast, the longer isoform uses the specific promoter upstream of the isolated 1-kilobase unit [5].

We have succeeded in getting one line of transgenic mice harboring the isolated 1-kilobase unit of the mouse cnpase gene and cre [6,7] (Fig. S1, Fig. S2). The mice were crossbred with ROSA26-β-galactosidase (ROSA26-BGAL, JAX's strain No. 003474) mice, identifying Cre recombinase-positive cells. We thus immunostained neonatal transgenic mouse brain tissues sliced vertically for myelinated axons in corpus callosum, which contains high-dense myelin sheaths. BGAL staining corresponded to the oligodendrocyte marker CC1, as well as to the oligodendrocyte precursor cell marker PDGFRβ (Fig. 1, A and B). We also sliced along an anterior and posterior axis in brain and performed BGAL staining (Fig. S3). Further studies allow us to understand not only its promoter activity but also studies on mechanisms underlying myelin development and diseases by using Cre mice.

Fig. S1

Generation of 46-kDa CNPase promoter-driven Cre recombinase transgenic mice. (A) I-digested transgene's linear construct expressing Cre recombinase is composed of enhancer sequence (Refs. 6 and 7), mouse 46-kDa CNPase promoter [5], Cre recombinase (derived from Addgene's Cre recombinase-containing plasmid No. 62730) containing nuclear localization signal, artificial intron (derived from TaKaRa Bio's pMEI5 vector), and human chorionic gonadotropin polyadenylation signal sequence. It was injected into C57BL/6 mouse fertilized eggs. (B) Presence of transgenes in founder mice was found using their tails (KAPA Mouse Genotyping Kit) by genomic PCR (5′-AATGCTTCTGTCCGTTTGCC-3′ and 5′-CTACACCAGAGACGGAAATC-3′ of 5′- and 3′- primers amplifying ~600 bps; 35 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min). Photographs of non-denaturing agarose gels indicated five mice harboring transgenes (lines 9, 17, 18, 19, 20, and 23). (C) Genomic PCR for -positive founder mice was further performed for the enhancer gene (5′-GAATATTAGCTAGGAGTTTCAGAAAGGGGGCCTG-3′ and 5′-ACTAGTGGGACTATGGTTGCTGACTAATTGAGATGC-3′ of 5′- and 3′-primers amplifying ~300 bps; 35 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 64 °C for 1 min, and extension at 72 °C for 1 min). Photographs of non-denaturing agarose gels also indicated five mice harboring transgenes (lines 9, 17, 18, 19, 20, and 23). From the results of the following tissue staining experiments, only the line 17 was positive for activities of the Cre recombinase at the detected levels. The offspring were used for experiments.

Fig. 1

Staining of β-Galactosidase (BGAL) and an oligodendrocyte marker in transgenic mouse corpus callosum. Transgenic mice of 46-kDa CNPase promoter-driven Cre recombinase were crossbred with ROSA26-BGAL mice. BGAL activities were detected in corpus callosum sections. In (A), BGAL (green) and CC1 (red, oligodendrocyte marker) were co-stained. In (B), BGAL (green) and PDGFRβ (red, oligodendrocyte precursor cell marker) were co-stained. Arrows indicate the representative colocalization positions of BGAL and an oligodendrocyte marker. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. S3

Mouse brain BGAL staining (46-kDa CNPase promoter-driven Cre recombinase transgenic mouse (line 17) x ROSA26-BGAL). Mouse whole brain at 7 days postnatal was sliced along an anterior and posterior axis and stained by detecting BGAL activities (blue). Stained lines were observed in olfactory bulb, corpus callosum, and hippocampus, consistent with myelin-rich positions in brain.

We have succeeded in getting one line of transgenic mice harboring the isolated 1-kilobase unit of the mouse cnpase gene and cre [6,7] (Figs. S1 and S2). The mice were crossbred with ROSA26-β-galactosidase (ROSA26-BGAL, JAX's strain No. 003474) mice, identifying Cre recombinase-positive cells. We thus immunostained neonatal transgenic mouse brain tissues sliced vertically for myelinated axons in corpus callosum, which contains high-dense myelin sheaths. BGAL staining corresponded to the oligodendrocyte marker CC1, as well as to the oligodendrocyte precursor cell marker PDGFRβ (Fig. 1, A and B). We also sliced along an anterior and posterior axis in brain and performed BGAL staining (Fig. S3). Further studies allow us to understand not only its promoter activity but also studies on mechanisms underlying myelin development and diseases by using Cre mice.

The following are the supplementary data related to this article.

Fig. S2

The nucleotide sequence of an enhancer, a mouse 46-kDa CNPase promoter, a Cre recombinase, and an intron. Double underlines indicate Cre recombinase's start and stop codons.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2018.03.003.