Generation of Genetically Modified Rats Using CRISPR/Cas9 Genome-Editing System to Reveal Novel Vitamin D Actions

Abstract

We previously revealed that the anti-proliferative activity of 25-hydroxyvitamin D3 (25(OH)D3) in human prostate PZ-HPV-7 cells depends on the direct action of 25(OH)D3 through the vitamin D receptor (VDR). We then attempted to confirm the direct action of 25(OH)D3 in vivo using Cyp27b1 knockout (KO) mice. Daily administration of 25(OH)D3 at 250 μg/kg bw/day rescued the rachitic conditions in Cyp27b1 KO mice. The plasma levels of calcium, phosphorus, and parathyroid hormonea as well as the bone mineral density and female sexual cycle were normalized via the administration of 25(OH)D3. These results strongly suggest the direct action of 25(OH)D3. However, to our surprise, normal levels of 1a,25(OH)2D3 were detected in the plasma of Cyp27b1 KO mice, probably due to Cyp27a1, which has a weak 1a-hydroxylation activity toward 25(OH)D3. Next, we generated a novel in vivo system using genetically modified (GM) rats deficient in the Cyp27b1 or Vdr gene to reveal the molecular mechanisms of vitamin D action. Human type II rickets model rats with mutant Vdr (R270L), which recognizes 1,25(OH)2D3 with an affinity equivalent to that of 25(OH)D3, were also generated. Cyp27b1-knockout (KO), Vdr-KO, and Vdr (R270L) rats showed symptoms of rickets, including growth retardation and abnormal bone formation. Among these model animals, Cyp27b1-KO rats had notably low levels of calcium in the blood and the most severe growth retardation, while Vdr-KO rats showed abnormal skin formation and alopecia. Administration of 25(OH)D3 restored rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. As shown in Cyp27b1-KO mice, 1,25(OH)2D3 was also synthesized in Cyp27b1-KO rats. In contrast, the effects of 25(OH)D3 on Vdr (R270L) rats strongly suggest that 25(OH)D3 exerts a direct action via VDR-genomic pathways. These results suggest that our novel in vivo system containing three different types of GM rats is useful for elucidating the molecular mechanism of vitamin D action.