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Manipulating transgenes using a chromosome vector.

Ikeno M, Suzuki N, Hasegawa Y, Okazaki T - Nucleic Acids Res. (2009)

Bottom Line: Expressive and repressive architectures of human STAT3 were established from naked DNA in mouse embryonic stem cells and CHO cells, respectively.Delivery of STAT3 within repressive architecture to embryonic stem cells resulted in STAT3 activation, accompanied by changes in DNA methylation.This technology for manipulating a single gene with a specific chromatin architecture could be utilized in applied biology, including stem cell science and regeneration medicine.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo, Japan. mikeno@fujita-hu.ac.jp

ABSTRACT
Recent technological advances have enabled us to visualize the organization and dynamics of local chromatin structures; however, the comprehensive mechanisms by which chromatin organization modulates gene regulation are poorly understood. We designed a human artificial chromosome vector that allowed manipulation of transgenes using a method for delivering chromatin architectures into different cell lines from human to fish. This methodology enabled analysis of de novo construction, epigenetic maintenance and changes in the chromatin architecture of specific genes. Expressive and repressive architectures of human STAT3 were established from naked DNA in mouse embryonic stem cells and CHO cells, respectively. Delivery of STAT3 within repressive architecture to embryonic stem cells resulted in STAT3 activation, accompanied by changes in DNA methylation. This technology for manipulating a single gene with a specific chromatin architecture could be utilized in applied biology, including stem cell science and regeneration medicine.

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Gene expression from the 25-4 vector. (a) Gene insertion into the 25-4 vector. The EGFP gene was inserted into 25-4 vector using Cre-lox recombination in HT1080 cells. Successful recombinants were selected by puromycin resistance (puro). (b) EGFP expression levels in 25-4. Fluorescence of CMV-, SV-, EF1- or cyclin A-driven expression of EGFP in HT1080 cells.
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Figure 3: Gene expression from the 25-4 vector. (a) Gene insertion into the 25-4 vector. The EGFP gene was inserted into 25-4 vector using Cre-lox recombination in HT1080 cells. Successful recombinants were selected by puromycin resistance (puro). (b) EGFP expression levels in 25-4. Fluorescence of CMV-, SV-, EF1- or cyclin A-driven expression of EGFP in HT1080 cells.

Mentions: To investigate the ability of the 25-4 vector gene insertion and expression system, we inserted an enhanced green fluorescent protein (EGFP) gene driven by several different promoters (CMV, SV, EF1 and cyclin A) by Cre-lox recombination of the lox66 site at the promoterless cassette and lox71 site in the gene expression cassette of 25-4 in HT1080 cells (Figure 3a). The major event in puromycin-resistant cell lines was insertion of the EGFP gene in one expression cassette in four sites of 25-4. EGFP expression was evaluated by fluorescence intensity (Figure 3b). The relative level of fluorescence of CMV-EGFP was greater than that of EF1-EGFP or SV-EGFP, and two orders of magnitude greater than that of cyclin A-EGFP. The expression of EGFP was homogeneous in >90% of cells in each line, indicating little variation in gene expression between cells within a clonal HT/25-4 cell line (Figure 3b). This promoter-dependent expression of EGFP from the 25-4 vector was also observed in NIH/3T3 and CHO cells.Figure 3.


Manipulating transgenes using a chromosome vector.

Ikeno M, Suzuki N, Hasegawa Y, Okazaki T - Nucleic Acids Res. (2009)

Gene expression from the 25-4 vector. (a) Gene insertion into the 25-4 vector. The EGFP gene was inserted into 25-4 vector using Cre-lox recombination in HT1080 cells. Successful recombinants were selected by puromycin resistance (puro). (b) EGFP expression levels in 25-4. Fluorescence of CMV-, SV-, EF1- or cyclin A-driven expression of EGFP in HT1080 cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665236&req=5

Figure 3: Gene expression from the 25-4 vector. (a) Gene insertion into the 25-4 vector. The EGFP gene was inserted into 25-4 vector using Cre-lox recombination in HT1080 cells. Successful recombinants were selected by puromycin resistance (puro). (b) EGFP expression levels in 25-4. Fluorescence of CMV-, SV-, EF1- or cyclin A-driven expression of EGFP in HT1080 cells.
Mentions: To investigate the ability of the 25-4 vector gene insertion and expression system, we inserted an enhanced green fluorescent protein (EGFP) gene driven by several different promoters (CMV, SV, EF1 and cyclin A) by Cre-lox recombination of the lox66 site at the promoterless cassette and lox71 site in the gene expression cassette of 25-4 in HT1080 cells (Figure 3a). The major event in puromycin-resistant cell lines was insertion of the EGFP gene in one expression cassette in four sites of 25-4. EGFP expression was evaluated by fluorescence intensity (Figure 3b). The relative level of fluorescence of CMV-EGFP was greater than that of EF1-EGFP or SV-EGFP, and two orders of magnitude greater than that of cyclin A-EGFP. The expression of EGFP was homogeneous in >90% of cells in each line, indicating little variation in gene expression between cells within a clonal HT/25-4 cell line (Figure 3b). This promoter-dependent expression of EGFP from the 25-4 vector was also observed in NIH/3T3 and CHO cells.Figure 3.

Bottom Line: Expressive and repressive architectures of human STAT3 were established from naked DNA in mouse embryonic stem cells and CHO cells, respectively.Delivery of STAT3 within repressive architecture to embryonic stem cells resulted in STAT3 activation, accompanied by changes in DNA methylation.This technology for manipulating a single gene with a specific chromatin architecture could be utilized in applied biology, including stem cell science and regeneration medicine.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo, Japan. mikeno@fujita-hu.ac.jp

ABSTRACT
Recent technological advances have enabled us to visualize the organization and dynamics of local chromatin structures; however, the comprehensive mechanisms by which chromatin organization modulates gene regulation are poorly understood. We designed a human artificial chromosome vector that allowed manipulation of transgenes using a method for delivering chromatin architectures into different cell lines from human to fish. This methodology enabled analysis of de novo construction, epigenetic maintenance and changes in the chromatin architecture of specific genes. Expressive and repressive architectures of human STAT3 were established from naked DNA in mouse embryonic stem cells and CHO cells, respectively. Delivery of STAT3 within repressive architecture to embryonic stem cells resulted in STAT3 activation, accompanied by changes in DNA methylation. This technology for manipulating a single gene with a specific chromatin architecture could be utilized in applied biology, including stem cell science and regeneration medicine.

Show MeSH