Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells

Abstract

A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage-fusion-bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.

Figure 1.

Strategy for amplifying a gene of interest on the MAC. (A) Amplification mechanism of IR/MAR plasmid in transfected cells suggested by our previous studies (5,7,10). The plasmid bearing IR/MAR (red arrow) is multimerized as a circular direct repeat in transfected cells that frequently recombines with DMs or any co-transfected DNA and can integrate into random sites in the chromosome arm; the repeat is further elongated by the BFB cycle in the chromosomal context. (B) Plasmid 6 has an HR2 sequence homologous to the MAC, an IR/MAR from the dihydrofolate reductase (DHFR) locus, and blasticidin resistance gene (BSR). The pSpCas9 HR2-2 plasmid expresses Cas9 protein and guides RNA specific to the center of the HR2 sequence; it has a puromycin resistance gene (puro). The two plasmids were co-transfected into MAC-bearing CHO DG44 cells (CD/M2 #44 cells). The photograph shows a FISH image where the MAC was detected by hybridizing Cot-1 DNA probe (red); chromosomes were counterstained with DAP (blue).

Figure 2.

Targeted amplification of the IR/MAR plasmid on the MAC. (A) Percentage of cells showing overlapping plasmid and MAC signals. (B–G) Representative FISH images of mouse Cot-1 probe specifically hybridizing to the MAC (red punctae and arrows) and hybridized plasmid probe (green punctae and arrows). DNA was counterstained with DAPI (blue). Insets in panels C–G show enlarged images.

Figure 3.

Mechanism of targeted amplification inferred from recombinant structure. (A–D) Plasmid–plasmid (A and B) or plasmid–MAC (C and D) recombination was analyzed by PCR using indicated primer sets flanking HR2, followed by agarose gel electrophoresis. Template DNA was isolated from untransfected CDM#44 (#44) cells, CDM#44 cells stably transfected with plasmid 6 (#44-6) or co-transfected with plasmid and pCas9 HR2-2 (#44-6 HR2-2), or clones (cl.) isolated from #44-6 HR2-2. (E) Model of the mechanism of targeted amplification inferred from the results of this study combined with the previous model of IR/MAR gene amplification (Figure 1A).

Figure 4.

Gene expression from amplified plasmid inside the MAC or normal chromosome. Cell clones were obtained from co-transfection of plasmid 6 and pCas9 HR2-2. For the logarithmically growing cells (But.−) or cells treated with 3 mM sodium butyrate for 3 days (But.+), green fluorescence from the d2EGFP gene product was analyzed by flow cytometry. Average fluorescence intensity is shown in each chart. Metaphase chromosome spreads from these clones were analyzed by dual-color FISH to detect the MAC (red) and plasmid (green); representative images are shown. For clone 1B3, each cell had three MACs (× 3) bearing the amplified plasmid.

Figure 5.

Visualization of MAC in live cells. (A) Metaphase chromosome spread from clone E5 cells analyzed by hybridizing plasmid (green) and mouse Cot-1 (red) probes. (B–D) Clone E5 cells grown in a glass-bottomed dish were fixed in paraformaldehyde and stained without (B) or with (C and D) DAPI. Differential interference contrast (DIC; gray), DAPI (blue) and LacR-GFP (green) images of logarithmically growing cells (B) or cells in prometaphase (C) or telophase (D) were obtained on a confocal microscope.

Figure 6.

BFB cycle on the MAC. (AandB) Metaphase chromosome spreads of clone E5 cells (A) or mouse embryonic fibroblasts (B) were hybridized with mouse major satellite probe (red); the plasmid probe (green) was simultaneously hybridized in (A) The BFB cycle in the normal chromosome arm (C) and the BFB cycle in the MAC (D) are depicted. Both cases involve dicentric chromosome formation; however, they differ in the site of dicentric breakage—the chromosome arm (C) and major satellite (D), respectively. The photograph that represents the resultant ladder structure is shown under each cartoon.