Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia.

Abstract

A minigene version of the human gene for type H procollagen (COI2A1) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened proa chains that associate with normal proa chains and thereby cause degradation of the shortened and normal proa chains through a process called procollagen suicide. Here we have prepared transgenic mice expressing a minigene version of the human COL2AJ gene. The design of the gene construct was based on several mutations in the genes for type I procollagen that produced in-frame deletions in codons for the repeating -Gly-Xaa-Yaa-amino acid sequence of the collagen triple helix and caused lethal variants of 01 (for reviews, see refs. 9 and 10). Because the collagen triple helix is formed by a series of hydrogen bonds and water bridges that link the -Gly-Xaa-Yaa-sequences in one proa chain to equivalent -Gly-Xaa-Yaa-sequences in the two other proa chains (see ref. 13), the presence of one shortened proa chain in a procollagen molecule can prevent folding into a stable triple helix and degradation of all three proa chains in a process referred to as "procollagen suicide" (11, 14, 15). MATERIALS AND METHODSGene Construct. The gene construct was prepared from a cosmid clone containing the human COL2AJ gene (ref. 16; kindly provided by Francesco Ramirez, Brookdale Center for Molecular Biology, Mt. Sinai Medical Center, New York). The insert in the cosmid was cleaved with Xba I, Sph I, and Cla I to generate four fragments ranging in size from 5 to 12 kilobases (kb) (Fig. 1). Three of the fragments were then assembled into a modified cosmid vector (17, 18) by four-way ligation. The insert was digested with Sal I, electrophoresed in an agarose gel, electroeluted, extracted with phenol/ chloroform/isoamyl alcohol (24:24:1), ethanol precipitated, and dissolved in 1 mM EDTA in 10 mM Tris HCl buffer (pH 7.4) for microinjection.Preparation of Transgenic Mice. One-cell stage mouse embryos (19) were obtained by mating of inbred FVB/N males and females. The partially deleted COL2AJ gene was microinjected into embryos at a concentration of 2 ,ug/ml with about 600 copies per embryo. Inbred CD1 females were used as the pseudopregnant recipients.Assay *To whom reprint requests should be addressed. 7640The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.

“…Attempts to understand the factors that may influence cartilage growth and development using transgenic technology have focused primarily on overexpressing mutated forms of type II collagen (Garofalo et al 1991;Vandenberg et al 1991, Metsaranta et al 1992. As expected, these mutations disturbed the assembly and processing of the homotrimeric type II collagen molecule in cartilage, resulting in chondrodysplasia.…”

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

“…Col2a1, which is expressed in differentiating chondrocytes, is essential for normal chondrogenesis in zebrafish and mammals (Vandenberg et al, 1991;Yan et al, 2002). Therefore, we examined whether the expression of col2a1 was altered in postmigratory neural crest cells of prep1.1 morphants.…”

Developmental pattern of expression of the mouse α1(XI) collagen gene (Col11a1)

“…INC paired development of mice harboring defective collagen genes. This large body of work includes transdominant overexpression of mutated versions of the genes coding for the al(I), al(II), al(IX), and al(X) collagen chain (Schnieke et al, 1983;Garofalo et al, 1991;Vandenberg et al, 1991;Nakata et al, 1993;Jacenko et al,, 1993); inactivation of the genes coding for the al(I), al(IX), and al(X) collagen chain (Stacy et al, 1988;Fassler et al, 1994;Rosati et al, 1994); and targeted mutagenesis of the gene coding for the a2(V) collagen chain . Additionally, the craniofacial and skeletal abnormalities characteristic of the mouse cho (for chondrodysplasia) have been recently associated with a frame-shift mutation in the gene coding for the a1 chain of type XI collagen (Colllal) (Li et al, 1995).…”