CORP: Using transgenic mice to study skeletal muscle physiology

Abstract

The development of tissue-specific inducible transgenic mice has provided a powerful tool to study gene function and cell biology in almost any tissue of interest at any given time within the animal's life. The purpose of this review is to describe how to use two different inducible transgenic systems, the Cre-loxP system and the Tet-ON/OFF system, that can be used to study skeletal muscle physiology. Myofiber- and satellite cell-specific Cre-loxP transgenic mice are described as is how these mice can be used to knockout a gene of interest or to deplete satellite cells in adult skeletal muscle, respectively. A myofiber-specific Tet-ON system is described as is how such mice can be used to overexpress a gene of interest or to label myonuclei. How to effectively breed and genotype the transgenic mice are also described in detail. The hope is this review will provide the basic information necessary to facilitate the incorporation of tissue-specific inducible transgenic mice into a skeletal muscle research program.

Keywords: Cre-loxP; Tet-ON/OFF; skeletal muscle; transgenic mice.

Fig. 1.

Myofiber-specific knockout of Dicer. MerCreMer [MCM; mutated estrogen receptor (Mer) fused on both the NH₂ and COOH terminal of Cre] is expressed under the myofiber-specific promoter [human α-skeletal actin (HSA)]. The MCM complex is sequestered in the cytosol by heat shock protein 90 (HSP90) and dissociates following the administration of tamoxifen. Once in the nucleus, the MCM recognizes, binds, and catalyzes the loxP sites, effectively removing the floxed DNA. In this example, the MCM excises exon 23 and 24 of the dicer DNA rendering a truncated protein, resulting in an inability to produce mature microRNAs.

Fig. 2.

Depletion of satellite cells in adult skeletal muscle using the paired box 7 (Pax7)-diphtheria toxin fragment A (DTA) mouse. The Pax7^CreER/CreER mouse was crossed to the Rosa26^DTA/DTA mouse to generate the Pax7-DTA mouse. Administration of tamoxifen to a Pax7-DTA mouse causes the dissociation of Cre estrogen receptor (CreER) from heatshock protein 90 (HSP90), allowing CreER to translocate to the nucleus where CreER binds to each loxP site and promotes recombination with the subsequent deletion of the upstream STOP cassette. Deletion of the STOP cassette allows transcription of the DTA cDNA, which causes apoptosis of satellite cells.

Fig. 3.

The Tet-OFF system. A tissue-specific promoter controls expression of the tetracycline transactivator (tTA). The tTA is composed of 2 elements, the tetracycline repressor (TetR) and the viral protein (VP16) transactivation domain; the VP16 transaction domain converts the TetR from a repressor to an activator of transcription. In the absence of doxycycline, the tTA remains bound to the tetracycline response element (TRE) and induces the expression of the downstream gene of interest. Under the administration of doxycycline, doxycycline binds to tTA causing its release from TRE and the loss of gene expression.

Fig. 4.

The Tet-ON system. A tissue-specific promoter controls expression of the reverse tetracycline transactivator (rtTA). The rtTA is composed of 2 elements, a mutated tetracycline repressor (TetR) and the viral protein (VP16) transactivation domain; the VP16 transaction domain converts the mutated TetR from a repressor to an activator of transcription. In the absence of doxycycline, the rtTA is unable to bind to the tetracycline response element (TRE). Upon doxycycline binding, rtTA is now able to the TRE and drive expression of the downstream gene of interest.

Fig. 5.

Myofiber-specific hybrid Tet-ON Cre-loxP system. One transgene contains the human α-skeletal muscle (HSA) promoter to drive myofiber-specific expression of the reverse tetracycline transactivator (rtTA). The second transgene contains a tetracycline response element (TRE) controlling expression of Cre. Upon doxycycline binding to the rtTA, the rtTA transcription factor is able to bind to the TRE and activate Cre transcription. This hybrid system allows for inducible Cre-mediated recombination without the use tamoxifen, providing an alternative strategy for studies investigating hormones and skeletal muscle. VP16, viral protein.

Fig. 6.

Breeding strategy to generate a myofiber-specific knockout mouse. Generating the human α-skeletal actin (HSA)-Dicer^f/f mouse, requires breeding a male, heterozygous HSA-MerCreMer (MCM) mouse crossed to a female, homozygous floxed Dicer (Dicer^f/f) mouse with 50% of the pups inheriting the HSA-MCM transgene and all pups inheriting one copy of the floxed Dicer allele, thus being heterozygous (Dicer ^f/+). A sexually mature (~8 wk) male HSA-Dicer ^f/+ mouse is then crossed to a homozygous Dicer^f/f female with 25% of the pups having the necessary genotype (HSA-Dicer^f/f) required to conditionally knockout Dicer in myofibers.

Fig. 7.

Breeding strategy to generate the paired box 7 (Pax7)-diphtheria toxin fragment A (DTA) mouse. Generating the Pax7-DTA mouse requires breeding a male, homozygous Pax7^CreER/CreER mouse to a female, homozygous Rosa26^DTA/DTA mouse. All the offspring from this cross will be heterozygous for Pax7^CreER/+ and Rosa26^DTA/+ alleles, designated Pax7-DTA. Upon tamoxifen administration, Cre-mediated recombination induces DTA expression resulting in satellite cell apoptosis.

Fig. 8.

Genotyping workflow. Workflow depicting the isolation of genomic DNA with downstream amplification by PCR to determine transgene heterozygosity.