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.

The daily rhythm of mice

“…It was found in a large-scale mutagenesis and phenotype screen from N-ethyl-N-nitrosourea treated mice. The mutation was mapped by positional cloning and its phenotype could be attenuated by the incorporation of additional transgenes containing a functional Clock locus into the genome of mice [7,8]. The mutation arose most probably from an A to T point mutation in the 5 0 splice site of intron 19.…”

“…This indicates that within the circadian oscillator homologous proteins can fulfill tissue-specific tasks of gene expression. Most probably, due to its nature as a dominant-negative regulator with normal DNA-binding capability, the mutant CLOCK D19 protein could interfere with the activity of both wild-type proteins and therefore provoke a striking phenotype [6][7][8].…”

BAC transgenic expression efficiency: bicistronic versus ATG‐fusion strategies

“…Unlike the knock-in approach, the BAC transgenic approach does not require the lengthy process of germline transmission and cross-breeding, and may even produce a stronger than knock-in expression of the transgene in mice with multiple copies of the transgene. Because of these benefits, the BAC transgenic technology has been increasingly applied to characterize the transcriptional regulation of gene expression (Reizis and Leder, 2001;Yu et al, 1999), label specific cell types (Chi et al, 2003), confirm the functionality of genetic mutations by in vivo complementation (Antoch et al, 1997;Probst et al, 1998), and reveal novel genetic functions (Heintz, 2000;Yang et al, 1999). Despite its expression fidelity and convenience, several issues are associated with this approach.…”

Efficient and precise engineering of a 200 kb β-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system

“…7,8 However, the lack of convenient techniques for performing genetic manipulations on BACs/PACs in the host E. coli DH10B strain imposes serious limitations for functional analysis. Homologous recombination in an F plasmid-based vector in E. coli was first used in 1989 to join overlapping Drosophila cosmid fragments to form a 125 kb fragment.…”

“…7,8 However, the lack of convenient techniques for performing genetic manipulations on BACs/PACs in the host E. coli DH10B strain imposes serious limitations for functional analysis.…”