Fig 1.
Generation of germline HA-hPIT1tg/+ transgenic mouse.
Scheme for recombination of the targeting vector pBT478.6-mKozak-CAG-LSL-HA-hPIT1 TARGATT into the H11 locus (A). Primer positions are shown, which were used in (B) to detect the modified H11attR site at 5’ junction (transgene inserted between attP1 and attP2) and 3’ junction (transgene inserted between attP2 and attP3) using primers from the Applied StemCell kit (primers 1,3 and 2,4, respectively). Germline excision shown in (C) was confirmed in (D) using primers detecting the wildtype H11 site (primers 896 and 897), the inactive LSL-positive (primers 879, 880) and the active germline ΔLSL alleles (primers 883 and 880). Primers 823 and 824 detect Cre recombinase, which is absent in the two mice shown consistent with germline excision of the LSL cassette of the mouse sample run in the left lane.
Table 1.
HA-hPIT1tg/+ mice are born at expected Mendelian rate.
Fig 2.
Expression of HA-hPIT1 and mPit1 in HA-hPIT1tg/+ and WT primary calvaria osteoblasts and various tissues.
(A) Semi-quantitative RT-PCR showing fold over actin expression in primary calvaria osteoblasts compared to wildtype at 80 days for hPIT1 and endogenous mouse Pit1 and 2 (mPit1 and 2). (B) Western blot of lysates obtained from PCOB from three separate HA-hPIT1tg/+ and one WT mouse using a lysate of HEK293 cells transfected with an expression plasmid encoding HA-hPIT1 as positive control, hybridized with anti-PIT1 rabbit polyclonal antibody (H-130, sc-98814, Santa Cruz Biotechnology) and detected with horseradish-peroxidase conjugated anti-rabbit IgG (mPit1 expected size 74 kDa, HA-hPIT1 expected size 75 kDa). (C) Semi-quantitative RT-PCR of total RNA from a HA-hPIT1tg/+ mouse shows expression of hPIT1 in different tissues (Liv = liver, K = kidney, C = colon, J = jejunum, I = ileum, B = brain, L = lung, T = testicle), compared to gastrocnemius (GC) and quadriceps (Q) from a wildtype (WT) mouse (n = 2) (D) Western blot of lysates from different tissues of a HA-hPIT1tg/+ and WT (hybridized with anti-PIT1 rabbit polyclonal antibody (H-130, sc-98814, Santa Cruz Biotechnology) and detected with horseradish-peroxidase conjugated anti-rabbit IgG) (mPit1 expected size 74 kDa, HA-hPIT1 expected size 75 kDa), (E) densitometric quantification of hPIT1 and mPit1 protein normalized to lung mPit1 and protein per lane from the Coomassie stains from two replicate Western blot experiments. (F) immunoprecipitation of HA-hPIT from quadriceps muscle obtained from a WT and HA-hPIT1tg/+ mouse. ****p<0.00002, ***p = 0.0002, **p = 0.002, *p = 0.03 vs. WT.
Fig 3.
Effect of HA-hPIT1 overexpression on biochemical parameters.
Biochemical parameters were determined in 80 day old mice and show aside from hyperphosphatemia no significant differences of means (A) and linear regression analysis (B) between WT and HA-hPIT1tg/+ mice. Means±SEM, n indicated by numbers inside bars, *p = 0.03 vs. WT.
Fig 4.
uCT analysis of P80 HA-hPIT1 transgenic mice.
Representative μCT images of two WT and HA-hPIT1tg/+ 80 day old males (A) and quantification of trabecular and cortical parameters (B) shows no significant effect of HA-hPIT1 overexpression. Means±SEM, n = 4 mice.