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. 2023 Dec 22;14:1009–1033. doi: 10.18632/oncotarget.28546

Figure 11. HAC-based gene delivery vector formation.

Figure 11

Step 1: Assembly of the 340 bp DNA dimers into 50–100 kb centromeric arrays by TAR cloning in yeast S. cerevisiae cells. TAR vector contains yeast and bacterial cassettes, a mammalian selectable marker (the Blasticidin resistance gene), and the 340 bp dimers as the targeting hooks. TAR vector linearized between the hooks and a mixture of dimers are transformed into yeast cells. End-to-end recombination of the DNA dimers followed by interaction of the recombined fragments with the vector hooks results in formation of the 50–100 kb centromeric arrays as circular YAC/BAC molecules. Step 2: TAR vector contains a mammalian selectable marker (the Blasticidin resistance gene) that allows a TAR/BAC clone containing a 100 kb centromeric array to be transferred to and maintained in human cells. Step 3: Formation of the HAC accompanied by input alphoid DNA multimerization up to ~1 Mb [141]. Step 4: A TAR-isolated gene of choice or an allelic variant of a gene (mut1, mut2 or mut3) is loaded into the single gene acceptor loxP site of the HAC by Cre-loxP mediated recombination. Step 5: Analysis of the mutant phenotypes of the gene.