The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

The CDK8 cyclin-dependent transcription-associated kinase and its less studied paralog, CDK19, regulate the expression of the dependant genes via several mechanisms. CDK8/19 can directly phosphorylate some transcription factors (ICN, STAT1), but at the same time these kinases being a component of the mediator complex regulate transcrition via interaction with chromatin in the promoter and enhancer regions of appropriate genes. Recently the papers have appeared showing that CDK8/19 has kinase-independent mechanisms of action through comparison of the effects of the kinase activity genetic inactivation and chemical inhibition. The study was aimed to generate transgenic mice capable of the induced and tissue-specific expression of the kinase-negative (showing no phosphorylation activity) form of CDK8, CDK8 (D173A), which could be later used to study the CDK8 kinase-independent mechanisms of action in vivo. We obtained four F0 transgenic animals by microinjection of linear DNA into the pronucleus, two of these animals became the ancestors of two distinct lines. The copy number of the integrated construct was measured for all F0 and the lines generated. This model may be used to study the kinase-independent properties of the CDK8/19 proteins.

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on alternative splicing and mutant exon skipping. Testing of such therapy requires animal models that carry mutations homologous to those found in human patients. Here, we report the generation of two genetically modified mouse lines, named “insT” and “insG”, with distinct mutations at the same position in exon 51 that lead to a frameshift, presumably causing protein truncation. Hemizygous males of both lines exhibit classical signs of muscular dystrophy in all muscle tissues except for the cardiac tissue. However, pathological changes are more pronounced in one of the lines. Membrane localization of the protein is reduced to the point of absence in one of the lines. Moreover, an increase in full-length isoform mRNA was detected in diaphragms of insG line mice. Although further work is needed to qualify these mutations as sole origins of dissimilarity, both genetically modified mouse lines are suitable models of DMD and can be used to test gene therapy based on alternative splicing.

Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations—one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (Flnc^AGA/AGA and Flnc^GA/GA) were embryonically lethal. We determined that Flnc^GA/GA embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (Flnc^GA/wt, Flnc^AGA/wt, and Flnc^GA/AGA). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (Flnc^GA/wt, Flnc^AGA/wt) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (Flnc^GA/AGA) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, Flnc^GA/AGA mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C.

Mouse models of human autoimmune diseases and inflammation are a challenging field because of the relatively low homology between the human and mouse immune systems. At the same time, inflammation plays a significant role in the pathogenesis of many diseases, strongly impacting quality of life and mortality. Cyclophilin A (CypA) is a pro-inflammatory factor, the ligand of immunosuppressive cyclosporin A, which mediates inflammation through multiple signaling pathways. Here, we describe a novel transgenic mouse model with Cre-dependent expression of the hPPIA gene in vascular endothelium and secretion of CypA into the bloodstream, which shows elevated blood levels of CypA upon activation. Being mostly asymptomatic under standard conditions, these mice exhibited more severe inflammation when provided with 3% dextran sulfate sodium solution instead of drinking water for 7 days. Inflammation symptoms precisely resembled those of ulcerative colitis and included deterioration of the colon crypts alongside the relatively normal duodenum. These results show that the elevated blood level of CypA enhances induced inflammation but does not cause inflammation by itself, suggesting its role in pro-inflammatory positive feedback loops and making CypA a suitable anti-inflammatory target. Moreover, our mouse strain is an applicable colitis model and can be used further in emerging inflammation research and testing anti-CypA targeted therapy.