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Genome-wide gene expression patterns in reprogramming MEF infected by SeVdp vectors carrying KLF4 WT or L507A mutant

ABSTRACT: Genome-wide gene expression patterns in reprogramming MEF infected by SeVdp vectors carrying KLF4 WT or L507A mutant

PROVIDER: PRJNA764699 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15972519_1.fastq.gz	Fastqsanger.gz
	SRR15972519_2.fastq.gz	Fastqsanger.gz
	SRR15972520_1.fastq.gz	Fastqsanger.gz
	SRR15972520_2.fastq.gz	Fastqsanger.gz
	SRR15972521_1.fastq.gz	Fastqsanger.gz

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Genome-wide gene expression patterns in reprogramming MEF infected by SeVdp vectors carrying KLF4 WT or L507A mutant

Project description:To evaluate global gene expression pattern changes between KLF4 L507A and WT during reprogramming, RNA-Seq was performed on the samples collected from reprogramming MEF infected with SeVdp vectors carrying KLF4 WT or L507A mutant. Original MEF and resulting miPSCs (mouse iPSCs) were also used as samples. RNA-seq library was generated using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) and NEBNext rRNA Delepletion Kit (New England Biolabs) by Lab Droid “Maholo” (Robotic Biology Institute) automatically. Paired-end sequencing of the libraries was performed using Novaseq 6000 SP Sequencing system using SP 100 cycle kit (Illumina). Reads from RNA-seq experiments were mapped to the mouse genome (GRCm38 or mm10) and analyzed for differential gene expression and PCA (principal component analysis) using CLC Genomics Workbench (Qiagen) software.

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Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)

Project description:Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO).

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Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)

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Genome-wide DNA binding patterns of KLF4 WT and mutant during reprogramming

Project description:On days 2 and 10 after transduction in an induction method, ChIP DNA samples were collected from reprogramming MEF transduced with SeVdp carrying a mutant or WT. ChIP-Seq was performed using an anti-human KLF4 antibody.

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Analysis of Klf4 factor stoichiometry effects during iPS cell derivation from mouse embryonic fibroblasts

Project description:Oct3/4, Sox2, Klf4, and c-Myc re-wire somatic cells to achieve induced pluripotency (iPS cells). However, subtle differences in reprogramming methodology may confound comparative studies of reprogramming-induced gene expression changes. We specifically focused on the design of polycistronic reprogramming constructs, which encode all four factors linked with 2A peptides. Notably, publically available cassettes were found to employ one of two Klf4 variants (Klf4S and Klf4L; GenBank Accession No’s: AAC52939.1 and AAC04892.1), differing only by nine N-terminal amino acids. In a polycistronic context, these two variants generated dissimilar protein stoichiometry, where Klf4L vectors produced more Klf4 protein than those encoding Klf4S. Employing piggyBac transposons, we systematically assessed gene expression changes during mouse embryonic fibroblast (MEF) reprogramming using a panel of novel and publically available polycistronic cassettes (Klf4S: OKMS, STEMCCA, EB-C5, WTSI; Klf4L: OSKM, MKOS, OK+9MS, OKN-HAMS). Strikingly, global gene expression patterns elicited by the panel of polycistronic cassettes at an intermediate stage of reprogramming (day6) diverged according to the Klf4 variant they encoded. Klf4L expressing vectors induced gene expression changes in MEFs similar to over-expression of Klf4 alone. Moreover, we found that these gene expression clusters were associated with differences in reprogramming hallmarks, including colony formation frequencies and the stabilization of transgene-independent pluripotency. Our data exposes a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, implicating a threshold in determining reprogramming outcomes. We hope this study will help to guide the comparison of compatible public gene expression data sets.

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Project description:In a pilot experiment to reprogramme MEF into endoderm, we infected MEF with the Yamanaka´s factors (O: Oct4, K: Klf4, S: Sox2, M:Myc), FoxA2 (F) and Gata4 (G). Global gene expression of isolated clones was performed.

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Project description:To further understand the mechanism of reprogramming, the mouse embryonic fibroblast cells were infected with Oct4, Klf4, c-Myc and Sox2 with special sequence. 1) Oct4 and Klf4 containing retrovirus was delivered on day 0. 2) Oct4, Klf4 and c-Myc were delivered on Day 1.5. 3) c-Myc and Sox-2 were deivered on Day 3. 4) Sox2 were delivered on Day 4.5. 5) Vitamin C containging medium were used from Day 6. The gene expression of Day 0 (MEF), Day 1.5, Day 3, Day 4.5 and Day 6 were analyzed with micro array to indentify the possible underlying mechanism. Especially the factors related to MET/EMT, cell cycle and epigenetic were focused. MEF cells were subjected for reprogramming with special protoocl: 1) Oct4 and Klf4 containing retrovirus were delivered on day 0. 2) Oct4, Klf4 and c-Myc were delivered on Day 1.5. 3) c-Myc and Sox-2 were deivered on Day 3. 4) Sox2 were delivered on Day 4.5. 5) Vitamin C containging medium were used from Day 6. RNA on Day 0 (MEF), Day 1.5, Day 3, Day 4.5 and Day 6 were collected for analysis.

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Expresion profile of MEF reprogrammed with YamanakaM-BM-4s factor together with FoxA2 and Gata4

Project description:In a pilot experiment to reprogramme MEF into endoderm, we infected MEF with the YamanakaM-BM-4s factors (O: Oct4, K: Klf4, S: Sox2, M:Myc), FoxA2 (F) and Gata4 (G). Global gene expression of isolated clones was performed. After infection of MEF with the YamanakaM-BM-4s factor (OKS/M), FoxA2 and Gata4, the cells were grown until colonies could be isolated and expanded independently. Global gene expression of several clones was performed.

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Genetic resistance elements carrying mef subclasses other than mef(A) in Streptococcus pyogenes.

Project description:In Streptococcus pyogenes, efflux-mediated erythromycin resistance is associated with the mef gene, represented mostly by mef(A), although a small portion of strains carry different mef subclasses. We characterized the composite genetic elements, including mef subclasses other than mef(A), associated with other resistance genes in S. pyogenes isolates. Determination of the genetic elements was performed by PCR mapping. The strains carrying mosaic mef(A/E), in which the 5' region was identical to mef(A) and the 3' region was identical to mef(E), also carried tet(O). The two genes were found enclosed in an element similar to S. pyogenes prophage Φm46.1, designated the Φm46.1-like element. In S. pyogenes strains carrying mef(E) and tet(M), mef(E) was included in a typical mega element, and in some strains, it was physically associated with tet(M) in the composite element Tn2009. S. pyogenes strains carrying mef(I) also carried catQ; the two genes were linked in a fragment representing a portion of the 5216IQ complex of Streptococcus pneumoniae, designated the defective IQ element. In the only isolate carrying a novel mef gene, this was associated with catQ and tet(M) in a genetic element similar to the 5216IQ complex of S. pneumoniae (5216IQ-like complex), suggesting that the novel mef is in fact a variant of mef(I). This study demonstrates that the composite elements containing mef are shared between S. pyogenes and S. pneumoniae and suggests that it is important to distinguish the mef subclass on the basis of the genetic element containing it.

| S-EPMC3122394 | biostudies-literature

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