Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

Abstract

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

Kidney Failure Alters Parathyroid Pin1 Phosphorylation and Parathyroid Hormone mRNA-Binding Proteins, Leading to Secondary Hyperparathyroidism

“…In some experiments, PT- EYFP (parathyroid-specific enhanced yellow fluorescent protein) mice were used to allow identification and microdissection of the mouse parathyroid glands for organ culture experiments. We generated the PT- EYFP mice by Cre-Lox recombination, mating PT- Cre mice, where the Cre recombinase is driven by the human PTH promoter (FVBTg[PT-Cre]; Jackson Laboratory, Bar Harbor, ME), 30 with R26-stop-EYFP/R26R-EYFP mice, where a loxP-flanked STOP sequence followed by the EYFP was inserted into the Gt(ROSA)26Sor locus 31 . Total DNA was extracted from tail samples of offspring and genotyping was performed by PCR.…”

“…We generated the PT-EYFP mice by Cre-Lox recombination, mating PT-Cre mice, where the Cre recombinase is driven by the human PTH promoter (FVBTg[PT-Cre]; Jackson Laboratory, Bar Harbor, ME), 30 with R26-stop-EYFP/R26R-EYFP mice, where a loxP-flanked STOP sequence followed by the EYFP was inserted into the Gt(ROSA)26Sor locus. 31 Total DNA was extracted from tail samples of offspring and genotyping was performed by PCR. The primers used were the following: for Cre: forward: 59-TGCCACGACCAAGTGACAGC-39 and reverse: 59-CCAG GTTACGGATATAGTTCATG-39 27 and for EYFP: ROSA 26R 59-AAAGTC GCTCTGAGTTGTTAT-39; BTG 60: 59-GAAAGACCGCGAAGAGTT TG-39; and BTG 62: 59-TAA GCCTGCCCAGAAGACTC-39.…”

Development of a Hypoparathyroid Male Rodent Model for Testing Delayed-Clearance PTH Molecules

“…The rodent model requires surgical parathyroidectomy or thyroparathyroidectomy (9,12,13). More recently a mouse model has been developed in which PTH glands can be highlighted using GFP for easy removal by surgery or for the targeted ablation of the PTH glands using diptheria toxin (14).…”