A new Col1a1 conditional knock-in mouse model to study osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) constitutes a family of bone fragility disorders characterized by both genetic and clinical heterogeneity. Several different mouse models reproduce the classic features of OI, and the most commonly studied carry either a spontaneous or genetically induced pathogenic variant in the Col1a1 or Col1a2 gene. When OI is caused by primary alterations of type I collagen, it represents a systemic connective tissue disease that, in addition to the skeleton, also affects several extra-skeletal tissues and organs, such as skin, teeth, lung, heart, and others, where the altered type I collagen is also expressed. Currently, existing mouse models harbor a disease-causing genetic variant in all tissues and do not allow assessing the primary vs secondary consequences of the mutation on a specific organ/system. Here, we describe the generation of the first conditional knock-in allele for Col1a1 that can express a severe OI-causing glycine substitution (p.Gly1146Arg) in the triple helical region of α1(I) but only after Cre-driven recombination in the tissue of choice. We called this new dominant allele Col1a1G1146R-Floxed/+ and introduced it into the murine model. We describe its validation by crossing mice carrying this allele with EIIA-Cre expressing mice and showing that offspring with the recombined allele reproduce the classic features of a severe form of OI. The new mouse model will be useful to study the tissue-specific impact of this severe mutation on organs, such as the lung, the heart, and others.

Keywords: bone; mouse models; osteogenesis imperfecta; skeleton; type I collagen.

Figure 1

Generation of the new mouse model. (A) Schematic diagram of the strategy for the generation of mice with the knock-in allele into the Col1a1 gene. The annotation of the various genetic elements is provided at the bottom of the figure. (B) Example of the identification of the four different mouse genotypes by PCR genotyping. (C) Comparison of body weight among male (n = 6-9) and female mice (n = 4-8) of the four genotypes of interest. p-values are indicated in the dot-plots and in red bold fonts when statistically different (one-way ANOVA).

Figure 2

X-ray and microCT imaging. (A) Representative X-ray imaging of male mice from the four different genotypes in prone position at 9 wk of age. The far-right panel is a blow-up of the mouse with the recombined allele and shows reduced size in addition to clear skeletal abnormalities and fractures, as labeled by arrows. (B) High-resolution microCT 3D renderings of mouse femurs from the four genotypes. Note how the femur from the mouse expressing the OI genetic variant is shorter and has drastically reduced trabecular bone.

Figure 3

DEXA analysis. BMD calculated from the whole body, the spine, and the femur in male (left panels) and female (right panels) mice at 9 wk of age (n = 6-9 and n = 3-8—one-way ANOVA).

Figure 4

MicroCT analysis of cancellous bone at the distal femur. Bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (conn-density), and apparent density in male mice at 9 wk of age (n = 6-9—one-way ANOVA). p-values that are statistically different are reported as such and in red fonts.

Figure 5

MicroCT analysis of cortical bone at the mid-shaft femur. (A) Cortical thickness (Ct.Th), polar moment of inertia, material density, and apparent density in male mice at 9 wk of age (n = 6-9—one-way ANOVA). p-values that are statistically different are reported in bold red fonts. (B) Representative 3D rendering of the bone cortical cross sections from the four genotypes.

Figure 6

Biomechanic assessment of the femur. (A) Extrinsic and intrinsic (B) parameters calculated from the 3 pt bending test of the femur in male mice at 9 wk of age (n = 6-8—one-way ANOVA). p-values that are statistically different are reported in red fonts.

Figure 7

Histology of the spine and lung. (A) Representative images of H&E stained, mid-coronal sections of lumbar vertebrae from the four mouse genotypes at 9 wk of age (scale bar = 1 mm). Average trabecular BV/TV and sample size for each genotype are reported below each panel. (B) H&E staining of lung sections from the four mouse genotypes at 9 wk of age, shown at two different magnifications (5×—scale bar = 500 μm, left panels; and 20×—scale bar = 100 μm, right panels). (C) Representation of the calculated MLI and the internal lung surface area (ISA) from the lung sections of 9-wk-old male mice (n = 3-5).