Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogs for their pharmacologic activities and potential therapeutic utility towards diseases of bone and mineral ion metabolism.  Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent versus human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such pre-clinical evaluations.  To overcome this potential uncertainty, we used a homologous recombination-based knock-in (KI) approach to generate a mouse (in host strain C57Bl/6N) in which cDNA encoding the human PTH1R replaces a segment (Exon 4) of the murine PTH1R gene such that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels.  The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least ten generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells.  The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogs in humans is reported here. 

PTH Peptides 

The peptides PTH(1-34) (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF), PTHrP(1-36) (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEI); LA-PTH(7-36) (LnLHQLdWKWIQDARRRAWLHKLIAEIHTAEI; nL = norleucine) or [Leu¹¹,dTrp¹²,Trp²³,Tyr³⁶]-PTHrP(7-36) (LLHDLdWKSIQDLRRRFWLHHLIAEIHTAEY; dW = d-tryptophan), were synthesized as C-terminal amides by the Massachusetts General Hospital Biopolymer Core facility. Peptides were purified by HPLC and confirmed for identity by mass spectroscopy. Abaloparatide (AVSEHQLLHDKGKSIQDLRRRELLEKLLbKLHTA; b = aminoisobutyric acid) was produced at Lonza (reference # 2AK1F). Peptides were dissolved in 10 mM acetic acid to a stock peptide concentration of 1.0 mM, and aliquots of the stock solutions were stored at -80˚C.

Generation of hPTH1R-KI Mice

Mouse lines were generated at Cyagen Biosciences Inc. (Santa Clara, Ca) using a homologous recombination-based knock-in strategy (see Figure 1). To engineer the targeting vector, homology arms were generated by PCR using BAC clone RP24-68N11 or RP23-278G23 from the C57Bl/6 library as template. The 5’ and 3’ homology arms contained sequences from intron 3 and intron 4 of the mouse PTH1R gene, respectively. Between the homology arms was inserted a segment of human PTH1R cDNA encoding from Val26 to the termination codon at codon 594 with an HA tag sequence (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) replacing the segment Tyr88-Glu96, followed by a rabbit poly adenylation transcriptional terminator sequence derived from the beta globin gene (rbgPA), and then a self-deleting neomycin resistance gene that is used for positive selection of ES cell clones and replaced by a single self-deleting anchor (SDA) sequence containing a LOX-P site upon ES cell selection. Splicing of the expressed transcript produced by the knock-in allele results in mouse exon 3 being joined at the codon corresponding to Leu25 in the mouse PTH1R to the codon for Val26 in the human cDNA sequence. Mouse exon 3 encodes mPTH1R residues Met1-Leu25, which includes the signal sequence, Met1-Ala22. Removal of the signal sequence during protein processing results in a mature PTH1R protein that is derived from the human cDNA sequence except for the mouse-derived segment of Tyr23-Ala24-Leu25, which is identical in the two species. The targeting vector also encoded diphtheria toxin A (DTA) used for negative selection and positive ES cell clones. The selected clones were injected into C57Bl/6N albino embryos, which were then re-implanted into CD-1 pseudo-pregnant females. Chimeric animals were identified by coat color, and germline transmission was confirmed by breeding with C57BL/6N females and subsequent genotyping of the offspring. Confirmed founder heterozygous targeted mice generated from ES cell clone 1A6 were used for further mating with C57BL/6N mice, and subsequent mating between heterozygous offspring yielded mice homozygous for the knock-in allele in the C57BL/6N background. Homozygous mice were transferred to and bred at Charles River Laboratories (Wilmington MA) and subsequently transferred to the Center for Comparative Medicine at Massachusetts General Hospital, where they were housed for the remainder of the study. Mice were maintained on a normal chow diet (ProLab IsoPRO RMH 3000, LabDiet St. Louis MO.) containing 1.1 % calcium and 0.8% phosphorus. All mouse procedures were approved by the MGH Institutional Animal Care and Use Committee.

DNA Sequence Analysis of the hPTH1R knock-in allele 

Genomic DNA was obtained from tail tissue from homozygous hPTH1R-KI mice and PCR was performed using the list of primers in Supplemental Table 1. PCR products were then submitted to the Massachusetts General Hospital Center for Computational and Integrative Biology DNA Core for Sanger sequencing analyses. The sequence of the entire insert with flanking regions of mouse Intron 3 and mouse intron 4 is displayed in Supplemental Figure 1. The locations in the DNA sequence of primers used for PCR and DNA sequence analysis are shown in Supplemental Figure 2. An alignment of the HA-PTH1R protein sequences encoded by the knock in allele and the mouse PTH1R is displayed in Supplemental Figure 2.

Western Blot Analysis of HA-hPTH1R in Kidney

Kidneys were isolated from two wild-type C57Bl/6N mice (WT-1, WT-2) and two homozygous hPTH1R-Ki mice (KI-1, KI-2) at 20 weeks of age, dissected on ice to remove the capsule, and placed in homogenization buffer (10 mM Tris-HCl, pH 7.8, supplemented with 1 mM EDTA, 1X-protease inhibitor cocktail (Bimake Inc. 100X, Reference # B14001), 1 mM DTT, 1 mM NaF, 0.2 mM Vanadate (New England Biolabs), 1% dodecylmaltoside (DDM) and 1 microM LA-PTH ^9,24. The tissue was homogenized using a Kimble Pellet Pestle Motor at 4˚C for 4 minutes. The homogenates were centrifuged at 1000xg for 10 min at 4˚C and the supernatants were collected and centrifuged at 14,000 xg for 30 min at 4˚C. The supernatants were removed, the pellets were resuspended in 600ul homogenization buffer, and the protein concentrations were determined by Bradford assay. The samples were then mixed with 2X Laemmli buffer, incubated at room temperature for 30 min, and after a brief storage at -80˚C, a sample volume containing 40 ug of protein was loaded onto an 8% acrylamide-SDS gel and after electrophoresis, the gels were processed for western blotting using an HRP-conjugated anti-HA mouse monoclonal antibody (BioLegend Cat# 901520, RRID:AB_2749912) diluted 1:500; the blots were treated with an HRP chemiluminescent ECL reagent (Thermofisher #34095), and images were acquired using an Azure Biosystems model C600 analyzer. After processing for HA-antibody binding, the blot was stripped and re-probed with an antibody for GAPDH (Cell Signaling Technology Cat# 2118, RRID: AB_561053) diluted 1:1000m followed by an HRP-conjugated goat anti rabbit IgG secondary antibody (Cell Signaling Technology Cat# 7074, RRID: AB_2099233), and imaged as for the anti-HA antibody.

Baseline Blood and Urine Biochemistry Analysis

Male and female hPTH1R-KI and wild-type C57Bl/6N control mice were euthanized at six months or at 13 months of age and cardiac blood was collected from the aorta using a 0.3 cc microinsulin syringe with a 31 g needle. The blood was placed into a plastic tube and centrifuged at 8,000 x g for 15 minutes at 4˚C and the supernatant (serum) was collected and placed into a new plastic tube and frozen at -80˚C. Urine was collected from the bladder using a 1.0 cc insulin syringe with a 25 g needle, placed into a plastic tube and frozen at -80˚C. Samples were thawed and an appropriate volume removed for assay. Assays were performed using colorimetric assay kits for: phosphate (AbCam. UK, Cat# ab65622); calcium (Stanbio Laboratory, Cat# 0150); creatinine (Stanbio Laboratory, Cat# 0430-500) and blood urea Nitrogen (Stanbio Laboratory, Cat# 0580); enzyme-linked immunoassay (EIA) kits for: C-terminal telopeptides of type I collagen (CTX-1; Immunodiagnostic Systems Cat# AC-06F1, RRID: AB_2801265); N-terminal propeptide of type I procollagen (PINP; Immunodiagnostic Systems Cat# AC-33F1, RRID: AB_2801263) and 1,25-Dihydroxy Vitamin D (Immunodiagnostic Systems Cat# AC-62F1, RRID: AB_2891249), and an ELISA kit formouse PTH(1-84) (Quidel Cat# 60-2305) containing antibodies for mouse PTH(53-84) (RRID: AB_2721077) and mouse PTH(1-12) (RRID: AB_2721076).

Micro-CT Analysis

Microcomputed tomography was performed on dissected femurs and skulls isolated from male and female wild-type C57Bl/6N and homozygous hPTH1R-Ki mice at six months and at 13 months of age using a desktop microtomographic imaging system (µCT 40, Scanco Medical AG, Brüttisellen, Switzerland). Samples were scanned with a 10-µm isotropic voxel size, 70 kV peak potential (kVp), 114µA X-ray tube intensity, and 300 ms integration time. Trabecular bone volume and microarchitecture were assessed in the intramedullary region of the distal femoral metaphysis beginning 0.5 mm superior to the growth plate and extending proximally for 1.5 mm (150 transverse slices); at the mid-shaft, analysis was performed on a 0.5 mm long region (50 transverse slices) to measure total area (Tt.Ar),cortical bone area (Ct.Ar), cortical bone area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct,Th), and cortical porosity. Data were analyzed according to gender, as differences between males and females within each genotype were significant (P < 0.05) for multiple parameters (see Table S-1).

Blood Calcium and Phosphorus Responses to PTH Peptide Injection

Changes in blood ionized calcium (Ca⁺⁺) and inorganic phosphorus (Pi) in response to PTH analog agonist peptide injections were assessed in homozygous hPTH1R-KI (8 female and 12 male) and wild-type C57Bl/6N (10 male and 10 female) mice at 9 weeks of age. hPTH1R-KI mice were injected subcutaneously with either vehicle (5 mM citric acid/150 mM NaCl/.05% Tween 80), or vehicle containing 40 nmol/kg of either PTH(1-34), PTHrP(1-36) or Abaloparatide (n=5 mice per group). For assessing calcemic responses, blood was collected via a small (~2 mm) tail vein puncture into heparinized capillary tubes (Multi-cap-S, Siemens Healthcare Diagnostics, reference #05656514) before injection (t=0), and 1, 2, 4 and 8 hours after injection, and analyzing the blood immediately using a Siemens RapidLab 348 Ca²⁺/pH analyzer. The calcemic response experiment was performed twice on two separate days for each set of 20 hPTH1R-KI and 20 wild-type control mice. For assessing changes in blood Pi in response to PTH ligand injection, hPTH1R-KI and wild-type mice (10 male and 10 female, each genotype) were injected with PTH(1-34), PTHrP(1-36) or Abaloparatide, each at a dose of 40 nmol/kg  (n=5 mice per group) and tail vein blood was collected into 1mL microcentrifuge tubes containing 3 uL 0.5 mM EDTA on ice at t=0, 1, 2, 4, and 8 hours post-injection. The blood samples were centrifuged at 8,000 rpm at 4oC for 15 minutes, and plasma supernatant collected and frozen at -80oC. Plasma phosphate was analyzed using a colorimetric phosphateassay kit (AbCam. UK, Reference # ab65622). For assessing responses to antagonist PTH analogs, WT or hPTH1R-KI mice were injected subcutaneously with either vehicle, vehicle containing PTH(1-34) alone at 40 nmol/kg, or with PTH(1-34) at 40 nmol/kg together with an antagonist peptide, LA-PTH(7-36) or [Leu¹¹,dTrp¹²,Trp²³,Tyr³⁶]-PTHrP(7-36), each at a dose of 500 nmol/kg (n=5 mice per group). Blood was collected from the tail vein and measured immediately for Ca⁺⁺ at varying time points, as described above for calcemic responses to PTH agonist injection. 

Data analysis

Data were processed using Microsoft Excel and Graphpad Prism version 9.0 software. Data are expressed as means ± SD or means ± SEM, as indicated in the figure legends and Tables, and were statistically analyzed by Student’s T test (two sample, equal variance); a P value of < 0.05 was inferred to be significant.