All data were presented as mean?��?SEM. For comparison of histomorphometric parameters in WT and KO mice, Student's t test was used. For quantitative analysis of immunostaining data, Student's t test was performed followed by chi-square test. Significance level was defined as p?<?0.05. To determine the role of LRP6 in osteoblastic bone formation, we generated mice lacking Lrp6 specifically in mature osteoblasts (Oc-Cre; Lrp6f/f, named KO hereafter) using Cre-mediated recombination. Transgenic mice expressing the Cre recombinase driven by the human osteocalcin promoter were crossed with homozygous mice that expressed loxP-flanked <a href="http://www.selleckchem.com/products/z-vad-fmk.html
Lrp6 (Lrp6f/f). To confirm that LRP6 deletion in KO mice was specific to osteoblasts, we performed PCR analysis using a combination of primers that specifically detected floxed Lrp6 alleles (Lrp6f) and null alleles (Lrp6��). Genomic DNA was extracted from skeletal tissues including calvariae, femur, and spine as well as several nonskeletal tissues such as brain, kidney, lung, liver, heart, and muscle from the KO mice. Cre-mediated recombination occurred exclusively in tissues that contained osteoblastic cells, whereas nonskeletal tissues retained the intact Lrp6-floxed Dolutegravir
alleles (Lrp6f) (Fig. 1A). LRP6-positive osteoblasts in femur tissue were also detected by immunohistochemical analysis of tissue sections using an antibody against LRP6. Although 58.6% of the bone surface osteoblasts expressed LRP6 in LRP6f/f mice (named WT hereafter), almost none of the osteoblasts expressed LRP6 in KO mice (Fig. 1B and Supplemental Fig. S1). No significant click here
reduction of LRP6-positive cell numbers in bone marrow was observed in KO mice compared with WT mice (Fig. 1C and Supplemental Fig. S1). Thus, LRP6 deficiency was limited to mature osteoblasts on the bone surface. KO mice were born at the expected Mendelian frequency, and all exhibited survival indistinguishable from that of WT mice. Mice expressing cre only did not exhibit any skeletal abnormality relative to wild-type mice in deferent studies,[34, 35] and no abnormal phenotype was observed in our analysis (data not shown). The body weight and femoral length were not decreased in either male or female KO mice compared with WT controls at 1 and 3 months of age (Table 1).