Fig 1.
Targeting strategy for LSL-TPP1 transgene.
CAG, chicken-actin promoter; Int, chicken actin splice donor, rabbit globin splice acceptor intron; STOP, stop cassette composed of PGK-neo / 4 x poly(A) sites / RNA polymerase pause site / linker containing stop codons in all reading frames; mTpp1, mouse TPP1 coding sequence; pA, rabbit globin poly(A) site.
Fig 2.
Testing LSL-TPP1 in CHO cells.
CHO cells were stably transfected with an AhdI fragment of pROSA26-TgLSL-TPP1 that contained only the CAG-LSL-TPP1 sequence. After selection with neo, CHO controls or cells containing CAG-LSL-TPP1 were transiently transfected with pCAG-cre and TPP1 activity measured at t = 0, 1 and 3 days. TPP1 activities were normalized to protein concentration and expressed as percentage of average CHO cell activity in the absence of CAG-LSL-TPP1. TPP1 activities in cells transfected with the transgene at 3 days were significantly elevated compared to all other conditions (p values ranging from 0.0003–0.0006 using Tukey’s multiple comparison test). All other comparisons had p values >0.7642.
Fig 3.
Where indicated, the stop element was constitutively removed from TgLSL-TPP1 mice by crossing with female animals containing the Zp3-cre transgene. A. Survival of animals expressing TgLSL-TPP1 before cre-mediated recombination (n = 56) or after (TgL-TPP1) (n = 28). Tpp1-/- (n = 1560) and Tpp1+/+ (n = 394) animals without transgene are historical controls. The ratio of median survival for animals with TgLSL-TPP1 (115 days) or controls without the transgene (132 days) was 0.8712 (95% CI 0.4716 to 1.61). B. Brain TPP1 activity in wild-type animals containing TgL-TPP1 (n = 3 animals per genotype). P value for significance between TPP1 activity in animals with or without transgene calculated using an unpaired two-tailed t-test was 0.0001. C. Survival of animals expressing TgL-TPP1 or littermates lacking the transgene in a Tpp1+/+ or Tpp1+/- background. Note that median survivals were undefined.
Fig 4.
Cre recombination in Tpp1-/- transgenic mice containing TgUBC-cre/ERT2.
Fig 5.
TPP1 activity in Tpp1-/- transgenic mice containing TgUBC-cre/ERT2 after tamoxifen-induced cre-recombination.
TPP1 activity in Tpp1-/- transgenic mice containing TgUBC-cre/ERT2 without tamoxifen treatment was compared with other cohorts by one way ANOVA with Tukey’s multiple comparisons test. In brain, there was no significant difference in TPP1 activity between Tpp1-/- animals with or without treatment, and no significant difference between Tpp1f/f and tamoxifen-treated Tpp1-/- animals with TgUBC-cre/ERT2. All other comparisons were significant with p values ranging from <0.0001 to 0.0088. β-Galactosidase activity was significantly lower in Tpp1f/f animals compared to Tpp1-/- animals with or without treatment and untreated Tpp1-/- animals with TgUBC-cre/ERT2 (p values ranging from 0.0065 to 0.0449) while there was no significant difference between other comparisons. In liver, there was no significant difference in TPP1 activity between untreated Tpp1-/- animals, treated Tpp1-/- animals and untreated Tpp1-/- animals with TgUBC-cre/ERT2. All other comparisons were significant with p values ranging from <0.0001 to 0.0021. β-Galactosidase activity was significantly lower in Tpp1f/f animals compared to Tpp1-/- animals without treatment and tamoxifen-treated Tpp1-/- animals with TgUBC-cre/ERT2 (p values ranging from 0.0016 to 0.0125) while there was no significant difference between other comparisons.
Fig 6.
Survival of Tpp1-/- mice in the presence of cre/ERT2 transgenes.
Note that animals were not treated with tamoxifen. Median survival and median survival ratio compared to Tpp1-/- littermate controls for each strain was: Tpp1-/- littermate controls (n = 161, 60 deaths), median survival 124 days; Tpp1-/- TgUBC-cre/ERT2 (n = 57, 14 deaths), median survival is 579 days, ratio to Tpp1-/- is 4.825 (95% CI 2.697 to 8.633); Tpp1-/- TgGFAP-cre/ERT2 (n = 54, 46 deaths), median survival is 117 days, ratio to Tpp1-/- is 0.975 (95% CI 0.664 to1.432); Tpp1-/- TgThy1-cre/ERT2 (n = 19, 4 deaths), median survival is 288 days, ratio to Tpp1-/- is 2.4 (95% CI 0.8723 to 6.604); Tpp1-/- Tgicre/ERT2 (n = 20, 19 deaths), median survival is 99 days, ratio to Tpp1-/- is 0.8208 (95% CI 0.49 to 1.375); Tppf/f (n = 754, 50 deaths), median survival is 639 days, ratio to Tpp1-/- is 5.325 (95% CI 3.659 to7.75).
Fig 7.
Testing tamoxifen-induced recombination of TgLSL-TPP1 by TgGFAP-cre/ERT2 and Tgicre/ERT2.
Six-week Tpp1+/- animals with indicated TgLSL-TPP1 and TgGFAP-cre/ERT2 (A) or Tgicre/ERT2 (B) genotypes were treated with tamoxifen as shown. Animals were killed one week after induction and assayed for TPP1 brain activity. C, 5-day old Tpp1-/- animals containing TgLSL-TPP1 with TgGFAP-cre/ERT2 were treated as indicated, killed one month after induction, and assayed for brain TPP1 activity. Tam, tamoxifen; Other transgene is TgLSL-TPP1 (TPP1 transgene with stop cassette) or TgL-TPP1 (active TPP1 transgene). The mean of each experimental condition was compared with every other mean by one-way ANOVA with p values corrected using Tukey’s multiple comparison test. No significant increase in TPP1 activity was detected under any experimental condition when animals containing both TgLSL-TPP1 and cre/ERT2 driver transgene were treated with tamoxifen compared to similar animals that were untreated or vehicle-treated.