title: Optimizing tamoxifen-inducible Cre/loxp system to reduce tamoxifen effect on bone turnover in long bones of young mice.
abstract: For tamoxifen-dependent Cre recombinase, also known as CreER recombinase, tamoxifen (TAM) is used to activate the Cre to generate time- and tissue-specific mouse mutants. TAM is a potent CreER system inducer; however, TAM is also an active selective estrogen receptor modulator (SERM) that can influence bone homeostasis. The purpose of this study was to optimize the TAM dose for Cre recombinase activation while minimizing the effects of TAM on bone turnover in young growing mice.
  
  
  METHODS
  To evaluate the effects of TAM on bone turnover and bone mass, 1-month-old wild-type male and female mice were intraperitoneally injected with TAM at 0, 1, 10 or 100mg/kg/day for four consecutive days, or 100, 300 mg/kg/day for one day. The distal femurs were analyzed one month after the last TAM injection by microCT, mechanical test, and surface-based bone histomorphometry. Similar doses of TAM were used in Col1 (2.3 kb)-CreERT2; mT/mG reporter male mice to evaluate the dose-dependent efficacy of Cre-ER activation in bone tissue.
  
  
  RESULTS
  A TAM dose of 100 mg/kg × 4 days significantly increased trabecular bone volume/total volume (BV/TV) of the distal femur, femur length, bone strength, and serum bone turnover markers compared to the 0mg control group. In contrast, TAM doses ≤ 10 mg/kg did not significantly change any of these parameters compared to the 0mg group, although a higher bone strength was observed in the 10mg group. Surface-based histomorphometry revealed that the 100mg/kg dose of TAM dose significantly increased trabecular bone formation and decreased periosteal bone formation at 1-week post-TAM treatment. Using the reporter mouse model Col1-CreERT2; mT/mG, we found that 10mg/kg TAM induced Col1-CreERT2 activity in bone at a comparable level to the 100mg/kg dose.
  
  
  CONCLUSIONS
  TAM treatment at 100mg/kg/day × 4 days significantly affects bone homeostasis, resulting in an anabolic bone effect on trabecular bone in 1-month-old male mice. However, a lower dose of TAM at 10 mg/kg/day × 4 days can yield similar Col1-CreERT2 induction efficacy with minimum effects on bone turnover in young male mice.
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA.
authors.forename: Zhendong A
authors.initials: ZA
authors.lastname: Zhong
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA.
authors.forename: Weihua
authors.initials: W
authors.lastname: Sun
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA; Department of Emergency Medicine The Second Xiangya Hospital of Central-South University, Changsha, Hunan, China.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA.
authors.forename: Yu-An E
authors.initials: YE
authors.lastname: Lay
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA.
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Lane
authors.affiliation: Center for Musculoskeletal Health, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA. Electronic address: yao@ucdavis.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Yao
mesh_terms: D000818:Animals; D016723:Bone Remodeling / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005269:Femur / Q000187:drug effects / Q000473:pathology; D055786:Gene Knockout Techniques / Q000379:methods*; D019426:Integrases; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020845:Selective Estrogen Receptor Modulators / Q000009:adverse effects / Q000494:pharmacology; D013629:Tamoxifen / Q000008:administration & dosage / Q000494:pharmacology*; D055114:X-Ray Microtomography
chemical_list: D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases
keywords: Bone formation; Bone turnover; Col1a1 (2.3kb); CreER; Estrogen; Tamoxifen