The targeting strategy allows the generation of a constitutive humanization of the Fkbp5 gene.
The targeting strategy is based on NCBI transcripts NM_010220_4 (mouse) and NM_001145775_2 (human).
Exon 2 contains the translation initiation codon. No cleavable signal peptide has been described for FKBP5 protein.
Mouse genomic sequence from the translation initiation codon in exon 2 to the termination codon in exon 11 has been replaced with its human counterpart.
Positive selection markers have been flanked by FRT (Neomycin resistance - NeoR) and F3 (Puromycin resistance - PuroR) sites and inserted into human intron 3 and intron 11, respectively.
The targeting vector has been generated using BAC clones from the mouse C57BL/6J RPCI-23 and human.
RPCI-11 BAC and/or CalTechD libraries and will be transfected into the Taconic Biosciences C57BL/6N Tac ES cell line.
Homologous recombinant clones will be isolated using double positive (NeoR and PuroR) and negative (Thymidine kinase - Tk) selections.
The humanized allele is obtained after in vitro Flp-mediated removal of the selection markers. The human FKBP5 gene will be expressed under the control of the endogenous mouse Fkbp5 promoter and should thus recapitulate the expression pattern of the mouse Fkbp5 gene. Replacement of the mouse Fkbp5 gene with its human counterpart results in loss of function of the mouse Fkbp5 gene.
The remaining recombination sites will be located in non-conserved regions of the genome.
Nold, V., Richter, N., Hengerer, B., Kolassa, I. & Allers, K. A. FKBP5 polymorphisms induce differential glucocorticoid responsiveness in primary CNS cells – First insights from novel humanized mice. Eur J Neurosci (2020) doi:10.1111/ejn.14999.