Dwarfism in homozygous Agc1^CreERT mice is associated with decreased expression of aggrecan

Abstract

SUMMARY Aggrecan (Acan), a large proteoglycan is abundantly expressed in cartilage tissue. Disruption of Acan gene causes dwarfism and perinatal lethality of homozygous mice. Due to sustained expression of Acan in the growth plate and articular cartilage, AgcCre model has been developed for the regulated ablation of target gene in chondrocytes. In this model, the IRES-CreERT-Neo-pgk transgene is knocked-in the 3′UTR of the Acan gene. We consistently noticed variable weight and size among the AgcCre littermates, prompting us to examine the cause of this phenotype. Wild-type, Cre-heterozygous (Agc+/Cre), and Cre-homozygous (AgcCre/Cre) littermates were indistinguishable at birth. However, by 1-month, AgcCre/Cre mice showed a significant reduction in body weight (18–27%) and body length (19–22%). Low body weight and dwarfism was sustained through adulthood and occurred in both genders. Compared with wild-type and Agc+/Cre littermates, long bones and vertebrae were shorter in AgcCre/Cre mice. Histological analysis of AgcCre/Cre mice revealed a significant reduction in the length of the growth plate and the thickness of articular cartilage. The amount of proteoglycan deposited in the cartilage of AgcCre/Cre mice was nearly half of the WT littermates. Analysis of gene expression indicates impaired differentiation of chondrocyte in hyaline cartilage of AgcCre/Cre mice. Notably, both Acan mRNA and protein was reduced by 50% in AgcCre/Cre mice. A strong correlation was noted between the level of Acan mRNA and the body length. Importantly, Agc+/Cre mice showed no overt skeletal phenotype. Thus to avoid misinterpretation of data, only the Agc+/Cre mice should be used for conditional deletion of a target gene in the cartilage tissue.

Aggrecan Hypomorphism Compromises Articular Cartilage Biomechanical Properties and Is Associated with Increased Incidence of Spontaneous Osteoarthritis

“…In our breeding colony, we have observed that, after two weeks of age, both male and female Agc1 CreERT2/CreERT2 mice were consistently smaller compared to wild type confirming the previously reported early postnatal dwarfism between one and three months [ 27 ]. We did not notice any other gross visual abnormalities up to one year of age: the Agc1 CreERT2/CreERT2 mice had apparently normal gait and physical activity.…”

“…In the Agc1 CreERT2 mouse model, the 3´ UTR of the aggrecan gene was disrupted by knocking in a Cre-ER T2 construct to generate an inducible, cartilage-specific deleter mouse line [ 16 ]. Although the original publication did not describe a phenotype, it has later been reported that Agc1 CreERT2/CreERT2 mice develop dwarfism by one month of age associated with shortening of the growth plate [ 27 ]. Now, here we show that the dwarf phenotype exists after the end of skeletal maturation having about 10–20% smaller skeletal elements of the Agc1 CreERT2/CreERT2 mice compared to wild type at 6 and 12 months of age ( Figure 1 ).…”

“…However, we have noticed during breeding of the line in our animal facility that the homozygous knock-in mice develop a postnatal dwarf phenotype. Indeed, it has been recently published that Agc1 CreERT2/CreERT2 mice have reduced body weight and length, and shorter endochondral bones compared to wild type and heterozygous mice [ 27 ]. Histological analysis indicated reduced height of the growth plate and the articular cartilage.…”

“…Histological analysis indicated reduced height of the growth plate and the articular cartilage. Further molecular and biochemical investigation revealed that the homozygous animals express about 50% of both aggrecan mRNA and protein in cartilage compared to the normal levels in wild type [ 27 ]. It was suggested that the decreased Agc1 expression may have been caused by the deletion of a 760 bp region of the 3’UTR, disrupting putative regulatory regions or by the negative effect of the targeting construct-derived pgk promoter on the activity of the endogenous Agc1 promoter [ 27 ].…”

“…Further molecular and biochemical investigation revealed that the homozygous animals express about 50% of both aggrecan mRNA and protein in cartilage compared to the normal levels in wild type [ 27 ]. It was suggested that the decreased Agc1 expression may have been caused by the deletion of a 760 bp region of the 3’UTR, disrupting putative regulatory regions or by the negative effect of the targeting construct-derived pgk promoter on the activity of the endogenous Agc1 promoter [ 27 ]. As a consequence of the disturbed skeletal growth in Agc1 CreERT2/CreERT2 mice, only heterozygous Agc1 CreERT2/+ mice were suggested to be used for conditional deletion of the floxed genes in chondrocytes [ 27 ].…”