Functional identification of the mouse circadian Clock gene by transgenic BAC rescue

Abstract

As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.

Figure 1

Physical Map of the Mouse Clock Locus Clock is localized to the mid-portion of mouse chromosome 5; SSLP markers D5Nwu2 and D5Mit307 define the nonrecombinant interval containing Clock. The relative positions of various sequence-tagged sites, SSLP markers, and restriction enzyme sites are indicated. D5Nwu2, D5Nwu1, D5Nwu14, D5Nwu7, and D5Nwu13 are SSLP markers described in King et al. (1997b). YAC55 appears to be a nonchimeric clone based on STS content mapping and long-range restriction mapping of overlapping YAC clones (data not shown). NotI, EagI, and NruI restriction profiles reveal two CpG islands. The three transcription units identified in this region, bendless, pFT27, and Clock, define the possible candidate genes; arrows indicate the direction of transcription and genomic extent, and introns and exons are indicated above the arrows. The BAC 54 rescuing clone is shaded. The two clones that fail to rescue, BAC 52 and the 100 kb NotI BAC 54 fragment, are shown as open bars. Telomeric (tel) and centromeric (cen) directions are indicated.

Figure 2

Transgene Copy Number Quantification BgIII-digested genomic DNA from transgenic and non-transgenic F1 progeny from each founder was probed with cDNA clone L4, which originates from the 3′untranslated region of the Clock gene. A 7kb fragment, which represents the endogenous Clock gene sequence) 1 copy/genome), was detected in all samples and was of much higher intensity in transgene-positive animals. A standard amount of BglII-digested BAC 54 DNA equivalent to 1, 3, and 5 copies per genome was mixed with 5 μg of control mouse DNA and loaded on the same gel. Band intensity was quantitated by densitometry, and transgene copy number was estimated (Table 1). (−) and (+) indicate mice negative or positive for transgene integration, respectively. The blot was normalized for DNA loading by hybridization with a mouse rhodopsin cDNA probe (a single copy copy gene [Al-Ubaidi et al., 1990]) (data not shown).

Figure 3

Genetic Cross Used to Produce Line TG36. Clock genotypes are depicted by shading, with wild-type open, heterozygotes shaded, and homozygotes closed. The presence of the 140kb BAC54 transgene is indicated by a double helix symbol. A Clock/+ transgenic founder male was bred with Clock heterozygous females so that mice of six genotypic classes (the three Clock genotypes, both with and without the transgene) were represented in the progeny.

Figure 4

The BAC 54 Transgene Rescues Mutant Circadian Phenotype and Affects Wild-Type Circadian Activity Wheel running activity records and period estimates are shown for F1 transgenic and control mice from transgenic line TG36, which carries the BAC 54 transgene. All animals were maintained on an LD 12:12 cycle (as indicated by the light/dark bar) for 5–7 days and transferred into constant darkness at a time indicated by the arrow. (A) Activity records of the wild-type mice with (right) and without (left) the BAC 54 transgene (tg). (B) Activity records of Clock/+ mice with (right) and without (left) the BAC 54 transgene. (C) Activity records of Clock/Clock mice with (right) and without (left) the BAC 54 transgene. (D) Histogram of period estimates for 83 F1 progeny from line TG36. Individual data points are indicated by open circles. Not all individual data points can be seen due to their overlap. Numbers on the bottom of the bars indicate the number of animals in each group that were wheel tested for their circadian behavior. Within this line, significant effects of Clock genotype (2 DF, F = 239, p < 0.00001), transgene presence (1 DF, F = 645, p < 0.00001), as well as genotype by transgene interactions (2 DF, F = 205, p < 0.00001) were detected (GLM ANOVA). Wild-type transgenic mice had significantly shorter periods than all non-transgenic groups, Clock/+ and Clock/Clock transgenic mice had significantly shorter periods than Clock/+ and Clock/Clock non-transgenic mice, and Clock/Clock non-transgenic mice had significantly longer periods than all other groups as determined by Tukey’s post hoc tests (p < 0.05).

Figure 5

The 100 kb BAC 54 and BAC 52 Transgenes Do Not Affect Circadian Phenotype Wheel running activity records and period estimates of Clock/+ F1 progeny from different BAC transgenic lines are shown. Both transgenic and control animals were behaviorally tested using the experimental protocol described in Figure 4 and the text. (A) Activity records of line TG55 Clock/+ progeny, in which the full-length BAC 54 was the transgenic construct. (tg) indicates transgenic animals’ records. (B) Activity records of line TG80 Clock/+ progeny, in which the 100 kb BAC 54 fragment was the transgenic construct. (C) Activity records of line TG121 Clock/+ progeny, in which the full-length BAC 52 was the transgenic construct. (D) Histogram of period estimates for 46 F1 animals from lines TG55, TG80, and TG121. Individual data points are indicated by open circles. Not all individual points can be seen due to overlapping values. Number of animals from each line tested is indicated on the bottom of the bar. Among the Clock/+ mice from these lines, significant effects of transgenic construct (2 DF, F = 9.60, p < 0.0005), transgene presence (1DF, F = 16.55, p < 0.0005), as well as construct by transgene interactions (2 DF, F = 10.8, p < 0.0001) were detected (GLM ANOVA). TG55 transgenic progeny had significantly shorter periods than all other groups as determined by Tukey’s post hoc tests (p < 0.05). There were no significant differences between transgenic and non-transgenic Clock/+ progeny in lines TG80 and TG121.

Figure 6

Clock mRNA Expression in Rescuing and Non-rescuing Lines Total RNA was isolated from the hypothalamic tissue of different BAC transgenic and control mice for Northern blot hybridization. Total RNA (25 μg) was hybridized with ³²P-labeled 51-base oligonucleotide complementary to 18S ribosomal RNA (C). RNA loading differs across the lines, as can be seen with the 18S ribosomal probe; however, the critical pairwise comparisons between non-transgenic (−) and transgenic (+) mice within each line can still be made. The BAC 54 transgenic lines TG14, TG36, and TG55 showed increased levels of Clock mRNA expression in transgene-positive mice, whereas the 100 kb BAC 54 line (TG97) and the BAC 52 line (TG121) showed similar levels of Clock mRNA between non-transgenic and transgenic mice. In this experiment, increased expression of Clock mRNA was not apparent in the BAC 54 line TG60 due to the low copy number of the transgene. Transgenic mice from the TG55 line also expressed a novel 2.4 kb transcript.

Figure 7

In Situ Hybridization Analysis of Clock mRNA in Line TG36 Coronal brain sections (20 μm) from TG36 Clock/Clock littermates, either lacking the transgene (A and B) or positive for the transgene (C and D), are shown. (A) and (C) are autoradiograms using antisense yz50probe, while (B) and (D) show the sense probe controls. Expression of Clock mRNA is widely distributed in the brains of both non-transgenic and transgenic animals. Highest levels of Clock mRNA were found in the suprachiasmatic nucleus (SCN) and pyriform cortex (PC). The transgene-positive animal (C) shows a selectively stronger signal in SCN as compared to Clock/Clock control animal (A). These representative sections are from the midportion of the SCN.