A congenic line of the C57BL/6J mouse strain that is proficient in melatonin synthesis

Abstract

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from nonfunctional alleles of the genes coding 2 key enzymes of the melatonin synthesis pathway: arylalkylamine-N-acetyltransferase (Aanat) and N-acetylserotonin-O-methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin-proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin-dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin-dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin-proficient B6 congenic line will be widely distributed.

Keywords: C3Heb/FeJ; C57BL/6J; CBA/CaJ; N-acetylserotonin-O-methyltransferase; arylalkylamine-N-acetyltransferase; circadian rhythms; congenic; melatonin; retina.

Figure 1

Identification and genotyping of alleles of the Aanat and Asmt genes in B6, C3H and CBA strains. A: Comparison of the sequences of Aanat and Asmt alleles. Locations of the sequences compared are indicated by the gene diagrams, where the exons are numbered. P: Pseudo-exon of the B6 type Aanat. Green: CBA sequence is identical to that of C3H but different from that of B6. Magenta: CBA sequence is different from those of C3H and B6. *: SNP used to design PCR primers. The two SNPs in the Aanat sequence (*, **) have been reported in B6 mice and the second one (**) shown to be responsible for the decreased AANAT enzymatic activity in this strain [7]. The boundaries of the pseudo-exon are exposed by the 2 arrows. B: Allele-specific PCR on the basis of SNPs was used to distinguish the different alleles. Visible PCR products represent the existence of a specific allele. The size of the PCR product is 284 bp for Aanat and 311 bp for Asmt.

Figure 2

Functional Aanat and Asmt alleles rescued nocturnal pineal melatonin biosynthesis in B6 congenic mice. Each dot represents an individual animal; horizontal short lines represent group means. Pineal glands were collected at CT19-CT20 in the dark. F1: double heterozygous for CBA alleles; N2*: maximum 1 CBA-type allele in either gene; N2**: double heterozygous for CBA-type alleles; N3: double homozygous for CBA-type alleles. The population variances are not significantly different [Brown-Forsythe test; F_(5,15) = 2.402 (P = 0.0778)] but the population means are significantly different [one-way ANOVA: F_(5,15) = 32.470 (P = 2.105E-8)]. Tukey post-hoc analysis indicates that CBA and N3 groups are not statistically different to one another (P > 0.5) but each is different from the other 3 groups (i.e. F1, N2*, and N2**) (P < 0.001); none of the F1, N2*, and N2** groups is significantly different from the 2 others (P > 0.05). n = 2-7.

Figure 3

Locomotor activity rhythms of CBA, B6 and congenic mice under a 12-h light/12-h dark cycle (12L/12D) or in constant dark conditions (D/D). A: Representative double-plotted profiles of wheel-running activity of a CBA mouse (left panel), a B6 mouse (middle panel), and a double-homozygous congenic mouse (right panel), obtained (from top to bottom) under a 12L/12D cycle, including after a 6-h phase shift (jetlag paradigm, black arrows), and D/D. B and C: Average period lengths of the wheel activity rhythm under 12L/12D (B) and D/D conditions (C). Mice from all genotypes had a period length of exactly 24.0 h under 12L/12D (B) and were able to run freely under D/D conditions with a period slightly shorter than 24 h (C). A two-way ANOVA of the data illustrated in (B) and (C) revealed significant differences between the lighting regimens (F_(1,18) = 129, P = 1.23e-9) as well as between genotypes (F_(2,18) = 25.6, P = 5.42e-6) and interaction lighting cycles x genotypes (F_(2,18) = 25.6, P = 5.42e-6). Tukey’s post hoc test revealed a significant difference between CBA and B6 mice (***, P = 2.92e-4) and CBA and congenic mice (**, P = 0.00679) but not between B6 and congenic animals (not significant [n.s.], P = 0.256). D: Time to adjust the onset of activity after a 6-h phase advance. A one-way ANOVA revealed significant differences between genotypes in the speed to which mice adjusted to the new phase (F_(2,9) = 76.5, P = 2.25e-6). Specifically, B6 mice adjusted much faster (within 2 days) to the new phase compared to CBA (***, P = 4.37e-6) or congenic mice (***, P = 4.27e-6), which did so in ~ 5 to 6 days. No difference was found between CBA and congenic mice (n.s., P = 0.246). Data were collected from 3 B6, 6 CBA, and 4 congenic mice. Error bars represent SEM. Shaded areas in A indicate periods of darkness.

Figure 4

Rhythmicity of melatonin biosynthesis and melatonin-dependent processes was restored in congenic animals. A: Subjective day/subjective night difference of pineal gland melatonin content in the N3 animals. Pineal glands were collected at CT6 (subjective day) and CT20 or CT21-22 (subjective night) and melatonin assayed by HPLC. Each dot represents an individual animal; group means are represented by short lines. *** P = 8.40 × 10⁻⁵, ** P = 0.004 (day vs CT21-22) or P = 0.008 (CT20 vs CT21-22), 2-tailed unpaired Student’s t-test. B: Subjective day/subjective night difference of retinal dopamine release in CBA, B6 and B6 congenic animals. Eyeballs were collected at CT6 (subjective day) and CT18-20 (subjective night) in the dark. Contents of DA and DOPAC were measured with HPLC-ECD. DA release was represented by DOPAC/DA ratio. Data are mean ± S.E.M. * P = 0.0175, *** P = 6.95 × 10⁻⁵, n.s. not significant, 2-tailed unpaired Student’s t-test. n = 4 for CBA and B6, at each time point and 6 for B6 congenic animals, at each time point.

Figure 5

Characterization of the 5′ and 3′ ends of the Aanat and Asmt genes. A. Visualization of PCR products of Aanat 5′ and 3′ ends amplified from CBA and B6 genomic DNA. Note the similar size of the PCR products of CBA and B6. B. comparison of CBA and B6 sequences for the Aanat 5′ and 3′ ends. SNPs are marked by asterisks. C. PCR product of 5′ end of CBA and B6 Asmt gene. Note the size difference of about 1 kb. D. Visualization of products of 2 PCR targeting 3′ end of Asmt gene using CBA and B6 genomic DNA. The PCR primers covered intron 7 for these 2 reactions. E. Visualization of products of PCR targeting 3′ end of Asmt gene using CBA and B6 genomic DNA. The PCR primers are both located inside Exon 8. F. Comparison of CBA and B6 sequences of 3′ end of the Asmt gene. A single SNP, marked with an asterisk, distinguish different alleles. For panels B and F, upper sequences are CBA and lower are B6.