Development and validation of triple knock-in humanized mice

PRIMARY OUTCOME

Establishment of functional humanized mouse model for SARS-CoV-2 research

EXPECTED OUTCOMES

- 1. Successful generation of homozygous triple knock-in mice with >95% targeting efficiency at all three loci, confirmed by sequencing and absence of off-target integration events - 2. Human-specific gene expression in relevant tissues: ACE2 in lung/kidney (5-10x mouse levels), TMPRSS2 in lung/prostate (3-7x mouse levels), FCGRT in multiple tissues (equivalent to mouse levels) - 3. SARS-CoV-2 susceptibility with viral loads of 10^6-10^8 copies/mg lung tissue at 2-4 days post-infection, compared to <10^3 copies/mg in wild-type controls - 4. Human-like antibody pharmacokinetics with IgG half-life of 8-12 days in TKI mice vs 1-2 days in wild-type mice, matching human t1/2 of 14-21 days when scaled for mouse physiology - 5. Stable germline transmission following Mendelian inheritance patterns with >90% breeding success rate and normal litter sizes (6-8 pups per litter) - 6. Normal baseline physiology with no significant differences in body weight, organ weights, or basic blood chemistry compared to wild-type controls - 7. Reproducible phenotype across multiple independent founders with <20% coefficient of variation in key functional readouts

SUCCESS CRITERIA

- • Genotypic validation: 100% correct integration at all three target loci confirmed by long-range PCR and whole genome sequencing with no off-target effects detected - • Expression validation: Human-specific gene expression detected by qRT-PCR with Ct values <30 and protein expression confirmed by Western blot with human-specific antibodies - • Viral susceptibility: >3-log difference in lung viral loads between TKI and wild-type mice (p<0.001, t-test with multiple comparisons correction) - • Antibody pharmacokinetics: IgG half-life >6 days in TKI mice vs <3 days in controls (p<0.01, n=6 per group, ANOVA followed by Tukey's test) - • Breeding performance: Stable colony establishment with >80% of breeding pairs producing viable offspring and consistent genotype ratios - • Phenotypic stability: <15% coefficient of variation in functional readouts across different breeding generations - • Reproducibility: Key findings replicated in at least 3 independent experiments with similar effect sizes

PROTOCOL

**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.

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