Cardiac-specific inducible and conditional gene targeting in mice

Abstract

Mouse genetic engineering has revolutionized our understanding of the molecular and genetic basis of heart development and disease. This technology involves conditional tissue-specific and temporal transgenic and gene targeting approaches, as well as introduction of polymorphisms into the mouse genome. These approaches are increasingly used to elucidate the genetic pathways underlying tissue homeostasis, physiology, and pathophysiology of adult heart. They have also led to the development of clinically relevant models of human cardiac diseases. Here, we review the technologies and their limitations in general and the cardiovascular research community in particular.

Figure 1. Cardiac cell types in adult heart

The heart is predominantly made up of myocardium, endocardium, cardiac fibroblasts, and epicardium. Epicardium is the outermost layer, whereas endocardium is the innermost layer. Both ventricular and atrial region are filled with myocardium and cardiac fibroblasts along with the coronary vasculature. The 4 cardiac valves (pulmonary, aortic, mitral, and tricuspid) contain valve interstitial cells, which are surrounded by valve endothelium. The valve annulus and vascular wall of the aorta and pulmonary trunk are predominantly made up of smooth muscle cells. The cardiac conduction system mainly consists of purkinje fiber and atrioventricular and sinoatrial nodes. All cardiac cell types are depicted in different colors and are labeled accordingly. RA indicates right atrium; TV, tricuspid valve; RV, right ventricle; LV, left ventricle; MV, mitral valve; LA, left atrium; AoV, aortic valve; PV, pulmonary valve. (Illustration credit: Cosmocyte/Ben Smith).

Figure 2. Cre and FLP recombinase systems

Cre and FLP recombinases recognize loxP (A) and FRT (B) sequences, respectively. These 34-bp sequences contain 13-bp inverted repeats flanking 8 bp unique sequence motifs that have directionality. When 2 loxP or 2 FRT sites line up a recombination occurs at the 8 bp motif. Depending on whether the 8-bp motifs are in the same or opposite orientation a deletion or insertion (C), inversion (D) or translocation (E) can occur.

Figure 3. Conditional gene targeting

A European Conditional Mouse Mutagenesis Program/Knockout Mouse Project gene targeting strategy that contains features for expression marking, removal of selectable marker gene, and conditional/ inducible gene inactivation. Homologous recombination results in “knockout (KO) first” alleles where gene function is ablated by a polyadenylation signal-mediated transcriptional stop at the end of the lacZ expression marker gene that is driven off the target gene promoter. Removal of the targeting cassette by a FLP recombinase germline deleter mouse leaves loxP sites flanking a “critical” exon resulting in mice harboring conditional/inducible-ready alleles. Alternatively, the original allele can be subjected to Cre recombinase to delete the critical exon which results in a lacZ-tagged, germline-null allele. SA indicates splice acceptor site; pA, poly A.

Figure 4. Inducible conditional gene targeting

A schematic representation of cardiac-specific conditional and Tam-inducible genetic deletion in mice. Spatiotemporally controlled cardiac-specific mutagenesis can be achieved by cardiac-specific expression of CreER^T2 in transgenic or knock-in mice. When cardiac-specific-CreER^T2 transgenic or knock-in mice are crossed to a mouse line carrying a conditional or floxed allele, the floxed gene is rapidly removed in response to Tam induction. In the absence of Tam, CreER^T2 is retained in the cytoplasm. Binding of Tam to the mutant ligand-binding domain of Cre-ER^T2 results in translocation of the Cre recombinase into the nucleus where it can recombine and delete floxed DNA in a conditional and Tam-inducible fashion. pA indicates poly A.

Figure 5. Tetracycline-inducible conditional gene targeting

The Tet system can be used to conditionally activate gene expression in the mouse. A, In Tet-ON system Dox-binding to reverse tetracycline controlled transactivator (rtTA) leads to transcriptional activation of transgene. B, In the Tet-OFF system the tTA binds to tetO and induces transcription in the absence of Dox. In the presence of Dox the transgene expression is prevented. C, The boxed region indicates another application of the Tet-inducible transgenic mouse technology. In this example, tissue-specific (TS) Cre recom-binase can be used to globally activate rtTA (Tet-ON) in R26r-rtTA knock-in mice. When these 2 mice are crossed to a tetO transgenic mice, and when the triple transgenic mice are subjected to Dox-induction, the transgene of interest can be expressed in a Dox-inducible fashion. tTA indicates tetracycline-dependent transactivator; Dox, doxycycline (ligand); tetO₇, Tet-responsive element.

Figure 6. Nuclease-mediated gene targeting

A, Diagrammatic representation of a pair of zinc finger nucleases (ZFNs) (top left) and transcription activator-like effector nucleases (TALENs) (top right) bound to DNA. Custom-made ZFNs and TALENs both have the FokI endonuclease fused to an array of zinc finger or transcription activator-like (TAL) effector (TALE) effector DNA-binding modules (gray triangles). Each ZFNs recognizes a 3-nucleotide sequence, whereas each of the DNA-binding modules of TALENs binds to a single nucleotide. ZFNs and TALENs arrays act as heterodimers, which create DNA cleavage at a user-specified genomic sequence. Zinc fingers (gray triangle) and TALENs (gray oval shapes) are shown with short vertical lines that indicate the main contacts of these nucleases with the DNA base pairs. FokI cleavage-encoding domains are shown as 2 large oval shapes (gray), with common cleavage sites, spaced by 4 bp, and indicated by vertical arrowheads. The spacer between the zinc finger or TALE binding sites is 6 bp, as indicated. In the presence of a homologous donor DNA (solid gray rectangular box, left), DNA repair can occur by homologous recombination using the donor as template. More recombination occurs near the original break, as indicated by the dark gray shading (right). The nonhomologous end joining results in mutations (eg, deletions, insertions, base substitutions) at the cleavage site (gray square, left). B, Scheme for producing gene-altered mice using nuclease-mediated gene targeting approach.

Figure 7. Mouse genetic engineering to manipulate alternative splicing in mice

Three exons (1–3) in a gene are shown. Three examples are provided to splice out or delete Exon 2 (black box). A, Exon 2 is floxed and can be deleted in conditional/inducible fashion. B, One STOP codon can be introduced along with the marker gene into the gene using random retroviral insertion or gene trap method. C, Depletion of isoforms containing Exon 2 by causing the destruction of the splice site. See text for details.