Figure 1
Schematic diagram outlining the construction of new MAC vectors. (1) Cloning of a mouse chromosome and construction of the 10MAC vector. Mouse embryonic fibroblasts (MEFs) containing the NeoR-tagged mouse chromosome 10 (mChr.10-NeoR) were fused with mouse A9 cells. The mChr.10-NeoR was transferred from whole cell hybrids into DT40 cells by microcell-mediated chromosome transfer (MMCT), and the DT40 microcell hybrids containing the mChr.10-NeoR were designated DT40 mChr.10-NeoR. Chromosome manipulation was performed in the homologous recombination-proficient DT40 microcell hybrids. The distal q-arm was deleted from the mChr.10-NeoR by telomere seeding-mediated chromosomal truncation. This mini-chromosome was designated as 10MAC. (2) Construction of the 10MAC1 vector and production of Tc mice. The plasmid containing EGFP and loxP-3’HPRT genes were cloned into a specific site of the 10MAC vector in DT40 cells by homologous recombination. The MAC vector containing EGFP and loxP site was designated as 10MAC1 vector. The EGFP can monitor the existence of 10MAC1 vector. The 10MAC1 was transferred into mouse embryonic stem (ES) cells via CHO cells for subsequent studies. To investigate the stability of the 10MAC1 vector, chimeric mice were produced from the ES cells containing the 10MAC1. The F1 mice were obtained by mating between chimeric and wild-type mice. (3) Construction of the 10MAC2 and 10MAC3 vectors and Cre/loxP-mediated gene loading. The 5’HPRT-loxP plasmid was targeted to a proximal region of the q-arm of the 10MAC vector in DT40 cells (10MAC2). The 10MAC2 vector was transferred into CHO (hprt−/−) cells and the circular vector carrying EGFP and loxP-3’HPRT was loaded into the 10MAC2 vector. The plasmid carrying EGFP and 5’-HPRT loxP was targeted to the MAC vector in DT40 cells (10MAC3). The 10MAC3 vector was transferred into CHO (hprt−/−) cells and the circular vector containing loxP-3’HPRT and tdTomato was loaded into the 10MAC3 vector.
