The FOLH1 gene, also known as prostate-specific membrane antigen (PSMA) or glutamate carboxypeptidase II (GCPII), encodes a type II transmembrane glycoprotein belonging to the M28 peptidase family. This protein functions as a glutamate carboxypeptidase, acting on various substrates including the nutrient folate (essential for its intestinal absorption) and the neuropeptide N-acetyl-l-aspartyl-l-glutamate (NAAG). In the brain, FOLH1 modulates excitatory neurotransmission by hydrolyzing NAAG, thereby releasing glutamate ^[1]. It is expressed in a wide range of tissues, including the prostate, central and peripheral nervous systems, kidney, small intestine, liver, and various tumor-associated vasculatures ^[2]. Aberrant expression of FOLH1 is notably associated with several diseases; it is significantly upregulated in prostate cancer cells and is a crucial diagnostic and prognostic indicator for this malignancy. Additionally, mutations in FOLH1 can lead to impaired intestinal absorption of dietary folates, resulting in low blood folate levels and hyperhomocysteinemia ^[3]. Its expression in the brain has also been implicated in pathological conditions related to glutamate excitotoxicity, and it is increasingly recognized as a therapeutic target in various solid tumors beyond prostate cancer, such as hepatocellular carcinoma, breast cancer, and Merkel cell carcinoma ^[2]. The B6-hFOLH1 mouse is a humanized model constructed by replacing aa.45 to partial intron 2 of the mouse Folh1 gene with the human FOLH1 cDNA of the extracellular domain (aa.44~750)-3’UTR of mouse Folh1-WPRE-BGH pA cassette. The murine cytoplasmic-transmembrane domain (aa.1~44) will be preserved. B6-hFOLH1 mice can be used for research into the pathogenesis of hyperhomocysteinemia and various solid tumors like prostate cancer, as well as for the screening, development, and safety evaluation of FOLH1-targeted drugs.

Grn is located on chromosome 11 of mice. Nuclease Technology was used to design sgRNA; Grn knockout mice were obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Ttbk2 is located on chromosome 2 of mice. SgRNA and ssDNA will be designed using Nuclease Technology; Ttbk2 conditional knockout mice will be obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm will be collected for cryopreservation.