pPH_pN_TK_pT_MHCK(d) was inserted into MAC4 using the Cre- loxP system. The TS-MAC was constructed using MAC4, which was derived from mouse chromosome 11, and contained EGFP and hygromycin resistance ( Hyg r) genes, a loxP site, and exons 1 and 2 of the hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene. Thus, the TS-MAC was correctly constructed in CHO cells and capable of transfer from the CHO cells to other targeted cells.įigure 1 Construction of a tumor-suppressing mouse artificial chromosome (TS-MAC). Fluorescence in situ hybridization (FISH) analysis showed that TS-MAC was maintained independently from CHO chromosomes ( Figure 1c, Table 2). PCR results of two representative clones, CHO/TS-MAC#1 and #2, and CHO/MAC4 as a negative control are shown in Figure 1b. Among them, 21 clones showed a positive result for all primer sets (data not shown). To confirm that the obtained CHO clones had the expected TS-MAC, polymerase chain reaction (PCR) analysis was performed with several primer sets ( Table 1). Therefore, the transfected CHO cells were expanded in HAT selection medium. CHO cells containing MAC4 that correctly recombined with the PAC vector could survive in medium containing hypoxanthine, aminopterin, and thymidine (HAT) ( Figure 1a). Thus, the PAC and Cre-recombinase expression vector were cotransfected in Chinese hamster ovary (CHO) cells containing MAC4 to mediate site-specific recombination. The PAC vector (pPH_pN_TK_pT_MHCK(d)) was designed for the MAC containing in vitro and in vivo safeguard systems, namely tumor suppressing-MAC (TS-MAC). These results suggest that the safeguard system is capable of suppressing tumor formation by the transplanted cells. Suppression of MHC H2-K(d)-expressing tumors in C57BL/6J mice was enhanced by immunization with MHC H2-K(d)-expressing splenocytes ( P < 0.01). The volume of tumors derived from B16F10 cells expressing allogenic MHC H2-K(d) was decreased significantly ( P < 0.01). Subcutaneous implantation of B16F10 cells into C57BL/6J mice resulted in high tumorigenicity. For proof-of-concept of the safeguard system, B16F10 mouse melanoma cells expressing the introduced H2-K(d) major histocompatibility complex (MHC class I)-allogenic haplotype were transplanted into recipient C57BL/6J mice expressing MHC H2-K(b). Here, we attempted to develop a tumor-suppressing mammalian artificial chromosome containing a safeguard system that uses the immune rejection system against allogeneic tissue from the host. ![]() Such safeguard systems should not disrupt the host genome and should have long-term stability. The development of a safeguard system to remove tumorigenic cells would allow safer clinical applications of stem cells for the treatment of patients with an intractable disease including genetic disorders. Gene Editing: Technology & Applications.
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