Phase 1: Mouse Strain Development -- Weeks 1-24
Generate triple knock-in mice using CRISPR-Cas9 technology targeting C57BL/6J background. Inject fertilized eggs with guide RNAs targeting mouse Ace2, Tmprss2, and Fcgrt loci along with donor templates containing human ACE2, TMPRSS2, and FCGRT sequences. Screen founder mice by PCR and Sanger sequencing. Breed founders to establish homozygous lines. Power analysis indicates n=20 breeding pairs minimum for establishing stable colonies.
...Phase 1: Mouse Strain Development -- Weeks 1-24
Generate triple knock-in mice using CRISPR-Cas9 technology targeting C57BL/6J background. Inject fertilized eggs with guide RNAs targeting mouse Ace2, Tmprss2, and Fcgrt loci along with donor templates containing human ACE2, TMPRSS2, and FCGRT sequences. Screen founder mice by PCR and Sanger sequencing. Breed founders to establish homozygous lines. Power analysis indicates n=20 breeding pairs minimum for establishing stable colonies.
Phase 2: Genotypic Validation -- Weeks 25-28
Perform comprehensive genomic validation of knock-in integration. Extract genomic DNA from tail biopsies using DNeasy Blood & Tissue Kit (Qiagen). Conduct long-range PCR across integration sites using Phusion High-Fidelity DNA Polymerase (ThermoFisher). Perform whole genome sequencing on 5 representative animals per line using Illumina NovaSeq platform. Validate absence of off-target effects through bioinformatics analysis of potential CRISPR sites.
Phase 3: Phenotypic and Expression Validation -- Weeks 29-36
Analyze gene expression levels using qRT-PCR with TaqMan probes specific for human sequences (n=12 mice per group, power=0.8, α=0.05). Extract RNA from lung, kidney, heart, and intestinal tissues using TRIzol reagent. Perform Western blotting for protein expression using anti-human ACE2 (Abcam ab108209), anti-human TMPRSS2 (Abcam ab92323), and anti-human FCGRT (Abcam ab193062) antibodies. Include wild-type C57BL/6J controls and human tissue positive controls.
Phase 4: Functional Validation - SARS-CoV-2 Susceptibility -- Weeks 37-42
Test viral susceptibility using SARS-CoV-2 WA1/2020 strain (10^5 PFU intranasal). Use n=8 mice per group (TKI vs wild-type controls) based on power analysis for detecting 2-log difference in viral loads. Sacrifice mice at 2, 4, and 6 days post-infection. Extract viral RNA from lung homogenates using QIAamp Viral RNA Mini Kit. Quantify viral loads by RT-qPCR targeting N gene. Perform plaque assays on Vero-E6 cells for infectious virus quantification.
Phase 5: Functional Validation - Antibody Pharmacokinetics -- Weeks 43-48
Administer human IgG1 antibodies (100 μg i.v.) to test FCGRT function. Use anti-RSV antibody palivizumab as test antibody (n=6 mice per group). Collect blood samples at 1, 6, 24, 72, 168, and 336 hours post-injection via submandibular bleeding. Measure antibody concentrations using human IgG-specific ELISA (Bethyl Laboratories). Calculate half-life using non-compartmental analysis in Phoenix WinNonlin. Compare to wild-type mice and published human pharmacokinetic data.
Phase 6: Breeding and Colony Establishment -- Weeks 49-52
Establish breeding colonies with documented genetic stability. Perform monthly genotyping of breeding animals. Maintain detailed breeding records and phenotypic monitoring. Validate germline transmission and Mendelian inheritance patterns. Prepare standard operating procedures for colony maintenance and distribute to research community.