Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

Gene targeting is an in situ manipulation of an endogenous gene in a precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. Conventional gene targeting has been achieved through the use of embryonic stem cells. However, such a procedure is often long, tedious, and expensive and has been limited in the mouse only due to a lack of usable embryonic stem cells in other species. This study was carried out to develop a much simplified procedure of gene targeting using E. coli recombinase recA and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene. The single-stranded oligonucleotide to target exon 3 of HPRT was 74 bases in length and included three phosphorothioate linkages at each terminus (also known as S-oligo) so as to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except for a single nucleotide that induces a mismatch between the introduced oligonucleotide and endogenous HPRT gene. Although the exact mechanism is yet unknown, endogenous repairing of such a mismatch would give rise to the conversion of TAT to TAG stop codon, thereby losing the function of the target gene. Prior to an introduction into zygotes, modified single-stranded oligonucleotides were preincubated with recA recombinase to enhance the homologous recombination. The recA-oligonucleotide complex was microinjected into the pronuclei of zygotes. Individual microinjected embryos that developed to the blastocyst stage were analyzed for the expected nucleotide conversion using PCR and subsequent sequencing. The conversion of TAT to TAG stop codon was confirmed in two embryos among forty tested blastocysts, so that the frequency of gene targeting was approximately 5%. The result suggests that the gene targeting was feasible by this relatively easier direct method. Subsequent transfer of gene-targeted embryos to recipients to obtain transgenic mice missing the function of HPRT gene is underway. Further technical refinement and enhancement of homologous recombination frequency will be required for the practical use of this new approach for gene targeting in mice.

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Genetic control of pluripotency epigenome determines differentiation bias in mouse embryonic stem cells

10.1101/2021.01.15.426861 ◽

2021 ◽

Author(s):

Candice Byers ◽

Catrina Spruce ◽

Haley J. Fortin ◽

Anne Czechanski ◽

Steven C. Munger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Genetic Regulation ◽

Embryonic Stem ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Embryoid Bodies ◽

Cell Fate Decisions

AbstractGenetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

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Regulation of the Nanog gene by both positive and negativecis-regulatory elements in embryonal carcinoma cells and embryonic stem cells

Molecular Reproduction and Development ◽

10.1002/mrd.20943 ◽

2009 ◽

Vol 76 (2) ◽

pp. 173-182 ◽

Cited By ~ 5

Author(s):

Brian Boer ◽

Jesse L. Cox ◽

David Claassen ◽

Sunil Kumar Mallanna ◽

Michelle Desler ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonal Carcinoma ◽

Embryonic Stem ◽

Regulatory Elements ◽

Carcinoma Cells ◽

Embryonal Carcinoma Cells ◽

Nanog Gene

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Targeted mutation of the Hprt gene in mouse embryonic stem cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.22.8583 ◽

1988 ◽

Vol 85 (22) ◽

pp. 8583-8587 ◽

Cited By ~ 128

Author(s):

T. Doetschman ◽

N. Maeda ◽

O. Smithies

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Hprt Gene ◽

Targeted Mutation

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Regulation of the myeloid-cell-expressed human gp91-phox gene as studied by transfer of yeast artificial chromosome clones into embryonic stem cells: suppression of a variegated cellular pattern of expression requires a full complement of distant cis elements.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2279 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2279-2290 ◽

Cited By ~ 22

Author(s):

L L Lien ◽

Y Lee ◽

S H Orkin

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Yeast Artificial Chromosome ◽

Embryonic Stem ◽

Myeloid Cells ◽

Artificial Chromosome ◽

Regulatory Elements ◽

Chromatin Domain ◽

Open Chromatin ◽

Cis Elements

Identifying the full repertoire of cis elements required for gene expression in mammalian cells (or animals) is challenging, given the moderate sizes of many loci. To study how the human gp91-phox gene is expressed specifically in myeloid hematopoietic cells, we introduced yeast artificial chromosome (YAC) clones and derivatives generated in yeast into mouse embryonic stem cells competent to differentiate to myeloid cells in vitro or into mouse chimeras. Fully appropriate regulation was recapitulated with a 130-kb YAC containing 60 and 30 kb of 5' and 3' flanking sequences, respectively. Immunodetection of human gp91-phox protein revealed uniform expression in individual myeloid cells. The removal of upstream sequences led to decreased overall expression which reflected largely a variegated pattern of expression, such that cells were either "on" or "off," rather than pancellular loss of expression. The proportion of clones displaying marked variegation increased with progressive deletion. DNase I mapping of chromatin identified two hypersensitive clusters, consistent with the presence of multiple regulatory elements. Our findings point to cooperative interactions of complex regulatory elements and suggest that the presence of an incomplete set of elements reduces the probability that an open chromatin domain (or active transcriptional complex) may form or be maintained in the face of repressive influences of neighboring chromatin.

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Synthetic and genomic regulatory elements reveal aspects of cis-regulatory grammar in mouse embryonic stem cells

eLife ◽

10.7554/elife.41279 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

Author(s):

Dana M King ◽

Clarice Kit Yee Hong ◽

James L Shepherdson ◽

David M Granas ◽

Brett B Maricque ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Embryonic Stem Cells ◽

Binding Site ◽

Binding Sites ◽

Embryonic Stem ◽

Regulatory Elements ◽

Genomic Sequences ◽

Primary Determinant ◽

Massively Parallel Reporter Assay

In embryonic stem cells (ESCs), a core transcription factor (TF) network establishes the gene expression program necessary for pluripotency. To address how interactions between four key TFs contribute to cis-regulation in mouse ESCs, we assayed two massively parallel reporter assay (MPRA) libraries composed of binding sites for SOX2, POU5F1 (OCT4), KLF4, and ESRRB. Comparisons between synthetic cis-regulatory elements and genomic sequences with comparable binding site configurations revealed some aspects of a regulatory grammar. The expression of synthetic elements is influenced by both the number and arrangement of binding sites. This grammar plays only a small role for genomic sequences, as the relative activities of genomic sequences are best explained by the predicted occupancy of binding sites, regardless of binding site identity and positioning. Our results suggest that the effects of transcription factor binding sites (TFBS) are influenced by the order and orientation of sites, but that in the genome the overall occupancy of TFs is the primary determinant of activity.

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Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-021-00563-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hee Jung Choi ◽

So Dam Jin ◽

Deivendran Rengaraj ◽

Jin Hwa Kim ◽

Bertrand Pain ◽

...

Keyword(s):

Stem Cells ◽

Transcriptional Regulation ◽

Embryonic Stem Cells ◽

Germ Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Regulatory Elements ◽

Cell Type ◽

Transcriptional Regulatory ◽

Cell Type Specific

Abstract Background NANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs. Results We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5′ flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG. Conclusions We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

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