Aim: Flvcr2 Conditional Knock-Out (cKO) and Reporter Knock-in (KI) by FLEx Strategy

•   The Flvc2GFP allele was generated by homologous recombination in embryonic stem cells. Two sets of inverted loxP sites (one regular set and one mutated set) direct recombination to remove the second exon and flip eGFP in-frame.
•   Validation of the cloned construct by Southern blot using two probes and two restriction enzymes.
•   To generate a knockout (null) allele, mice carrying a Cre transgene expressed under the control of the Mef2c promoter were crossed with the floxed mice. Upon Cre-mediated recombination, the second exon was removed, leading to the inactivation of the gene and the expression of GFP under the control of the endogenous locus.
•   Flvcr2 expression, as indicated by GFP reporter analysis, was prominent in the brain, with notable signals also detected in lung alveolar macrophages and sporadic expression in intestinal epithelial cells and hepatocytes in adult Flvcr2+/GFP mice.
•   Flvcr2 conditional knock-out and GFP knock-in mice demonstrate that Flvcr2 was necessary for angiogenic sprouting in the brain, but dispensable for maintaining the blood-brain barrier. This model provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.

Publication:

Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier

Santander N, Lizama CO, Meky E, McKinsey GL, Jung B, Sheppard D, Betsholtz C, and Arnold TD (2020) Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier. J Clin Invest, 130, 4055-4068.

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Aim: Flvcr2 Conditional Knock-Out (cKO) and Reporter Knock-in (KI) by FLEx Strategy

• •   Allele Design & Validation: The Flvc2GFP allele was generated by homologous recombination in embryonic stem cells. Two sets of inverted loxP sites (one regular set and one mutated set) were introduced in order to remove the second exon and flip eGFP in-frame upon Cre recombination. Validation of the cloned construct by Southern blot was achieved using two probes and two restriction enzymes.
    
    
  •   Model Validation: To generate a knockout (null) allele, mice carrying a Cre transgene expressed under the control of the Mef2c promoter were crossed with the floxed mice. Upon Cre-mediated recombination, the second exon was removed, leading to the inactivation of the gene and the expression of GFP under the control of the endogenous locus. Flvcr2 expression, as indicated by GFP reporter analysis, was prominent in the brain, with notable signals also detected in lung alveolar macrophages. Sporadic expression was observed in intestinal epithelial cells and hepatocytes in adult Flvcr2+/GFP mice.
    
    
  •   Biologic Results: Studies in Flvcr2 conditional knock-out/GFP knock-in mice demonstrated that Flvcr2 was necessary for angiogenic sprouting in the brain, but dispensable for maintaining the blood-brain barrier. This model provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
    
    

Publication:
Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier

Santander N, Lizama CO, Meky E, McKinsey GL, Jung B, Sheppard D, Betsholtz C, and Arnold TD (2020) Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier. J Clin Invest, 130, 4055-4068.

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Aim: Microglial P2y12 Receptor Conditional Knock-Out (cKO) by CRISPR/Cas9-based Extreme Genome Editing (EGETM) System

·   Allele Design & Validation: Guide RNAs (gRNAs) were designed to target the regions upstream of exon 4 and downstream of the 3’UTR of P2ry12. Exon 4 was flanked with one loxP fragment inserted into intron 3 and the other was inserted downstream of the 3’UTR. P2Y12-floxed mice were then crossed with the CX3CR1Cre/+ mice to obtain P2Y12f/f;CX3CR1Cre/+ (conditional KO) mice. Upon Cre-mediated recombination, exon 4 was removed, leading to the inactivation of the gene. Immunostaining results showed that P2Y12R expression was completely removed from the conditional KO mice in the brain of adult mice.
·   Biologic Results: P2y12 receptor deficiency increased seizure severity in P2Y12f/f;CX3CR1Cre/+ mice compared to WT mice. The results from microglial P2y12 receptor conditional knock-out mice indicate that microglia enhance epileptogenesis and targeting microglia in general or microglial P2Y12R in specific to ameliorate proepileptogenic processes could be a novel therapeutic strategy in the clinic.

Figure 1. Targeting strategy for conditional knockout mice.

Publication:

Microglial P2Y12 receptor regulates ventral hippocampal CA1 neuronal excitability and innate fear in mice

Peng J, Liu Y, Umpierre AD, Xie M, Tian DS, Richardson JR, and Wu LJ (2019) Microglial P2Y12 receptor regulates ventral hippocampal CA1 neuronal excitability and innate fear in mice. Mol Brain, 12, 71.

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Aim: hCD63-GFP Conditional Knock-in (cKI) in Rosa26 ‘Safe Harbor’ Locus

·    Allele Design & Validation: hCD63-GFPf/f knock-in mice were generated by homologous recombination in embryonic stem cells. A CAG promoter, loxP-floxed stop codon, and human CD63-copGFP-6xHis cassette were inserted into the Rosa26 ‘safe harbor’ locus. The hCD63-GFPf/f mice were crossed with tamoxifen-inducible Cre mice under the control of the CaMKII promoter to generate the CaMKII-CreERTCD63-GFPf/+ mice. After 4-hydroxytamoxifen (4-OHT) injection and Cre-mediated recombination, the stop codon was removed, leading to the expression of hCD63 with a GFP tag under the control of the CAG promoter.
·   Model Validation: AAV8-CaMKII-Cre_mCherry virus particles from which mCherry-tagged Cre is expressed under the CaMKII promoter were focally injected into the motor cortex of CD63-GFPf/+ mice. Both CD63-GFP and mCherry signals are observed in the proximal section of the injection site.
·   Biologic Results: The Rosa26-hCD63-GFP conditional knock-in mice demonstrated that neuronal exosomes can be secreted and transferred into astrocytes in vivo. This finding potentially opens a new frontier in understanding CNS intercellular communication. 

Publication:
Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS

Men Y, Yelick J, Jin S, Tian Y, Chiang MSR, Higashimori H, Brown E, Jarvis R, and Yang Y (2019) Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS. Nat Commun, 10, 4136.

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B-hTFR1 mice

Aim: Generation of a humanized TFR1 mouse model

• Introduction: Transferrin receptor 1 (TFR1), also known as CD71, is a cell surface receptor that plays a major role in cellular iron uptake via receptor-mediated endocytosis. The efficient cellular uptake properties of TFR1 have been exploited for site-specific delivery of anti-cancer drugs and biologics to malignant cells overexpressing TFR1.
  
  Allele Design & Validation: A humanized B-hTFR1 mouse model for evaluating TFR1-targeted therapeutics in preclinical studies was successfully generated at Biocytogen. In this model, exons 4-19 of the mouse Tfr1 gene that encodes the receptor’s extracellular domain were replaced by human counterparts (Figure 1). Protein expression analysis by flow cytometry in erythroid cells isolated from bone marrow showed that mouse Tfr1 was detectable in WT mice and human TFR1 was exclusively detectable in homozygous B-hTFR1 mice. Similarly, immunofluorescence staining in brain samples indicated that human TFR1 was exclusively detectable in the microvascular endothelium of homozygous B-hTFR1 mice.

   

  Biologic Results: Anti-human TFR1 bispecific antibody was successfully detected in brain tissues of the B-hTFR1 mouse after intravenous administration of the antibody, suggesting a robust uptake of the antibody via the hTFR1 receptor.

Figure 1. Targeting strategy for the generation of humanized TFR1 mice.

B-hTFR1 mouse model product page