Figure 2.

Construction of a complex, propagated artificial chromosome in C. elegans using fragmented yeast genomic DNA and marker genes. (A) A schematic map of the co-injection markers, NeoR, gfp::h2b, and mCherry (NGM), PCR-amplified from plasmid WYYp228. The purified PCR product (0.5 ng/μl) was used in the co-injection mix with fragmented yeast genomic DNA (150 ng/μl). The antibiotic resistance gene (NeoR) is driven by the ubiquitous rps-27 promoter. gfp::h2b is driven by the germline mex-5 promoter, and mCherry is driven by the somatic, body wall muscle myo-3 promoter. The scale bar (in bp) is shown. (B) A schematic diagram of the experimental approach used to generate and select a complex, propagated artificial chromosome in C. elegans for downstream whole-genome sequencing and chromatin immunoprecipitation followed by sequencing analysis (ChIP-seq). (C) Quantification of a unique endogenous locus on Chromosome II (9565795:9565963), mCherry marker, and yeast rDNA copy number in strains (L2.1 and L2.2) with an AC by quantitative PCR. L2.1 and L2.2 are different F2 progenies from the same F1 produced by an injected worm. Genomic DNA was obtained from dauer worms on starved plates. The gene copy number is normalized to the unique endogenous locus, which is assumed to have two copies (before DNA replication) in the diploid organism. Error bars indicate 95% confidence interval (CI) for the mean. (D) Representative marker gene expression in embryos and adult worms from strain L2.1. (E) The propagated AC (yellow arrowhead) was stained in oocytes and embryos by FISH probes made from yeast genomic DNA. The AC, often smaller than the endogenous chromosomes, lacks AIR-2 signal in the oocytes. DNA was stained by DAPI. In the multi-cell embryos, the propagated ACs aligned at the metaphase plate and segregated with endogenous chromosomes in anaphase and telophase. Scale bar represents 2 and 5 μm in the oocytes and embryos, respectively.