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Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

ABSTRACT: Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

PROVIDER: PRJNA429696 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7227480.fastq.gz	Fastqsanger.gz
	SRR7227490.fastq.gz	Fastqsanger.gz
	SRR7227500.fastq.gz	Fastqsanger.gz
	SRR7227510.fastq.gz	Fastqsanger.gz
	SRR7227481.fastq.gz	Fastqsanger.gz

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Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

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Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

Project description:The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system (E-BACs) consisting of ∼200 kb human bacterial artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) stable segregation elements. E-BACs were stably maintained as autonomous mini-chromosomes in EBNA1-expressing HeLa or human induced pluripotent stem cells (hiPSCs) and established distinct RT patterns. An E-BAC harboring an early replicating chromosomal region replicated early during S phase, while E-BACs derived from RT transition regions (TTRs) and late replicating regions replicated in mid to late S phase. Analysis of E-BAC interactions with cellular chromatin (4C-seq) revealed that the early replicating E-BAC interacted broadly throughout the genome and preferentially with the early replicating compartment of the nucleus. In contrast, mid- to late-replicating E-BACs interacted with more specific late replicating chromosomal segments, some of which were shared between different E-BACs. Together, we describe a versatile system in which to study the structure and function of chromosomal segments that are stably maintained separately from the influence of cellular chromosome context.

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Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

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Project description:Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vector, e.g., episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown construct (see Kazuki et al 2011). The iHAC1 partially reprogrammed MEFs, and the iHAC2 efficiently reprogrammed MEFs. Global gene expression showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. We established iHAC-free iPS cells by isolating cells that spontaneously lost the iHAC2. Analyses of pluripotent markers, teratomas, and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex Virus Thymidine Kinase were eliminated by Ganciclovir treatment; therefore, the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a safeguard system.

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