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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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Transfer of human STAT3 chromatin architecture. (a) Comparison of human, mouse and Chinese hamster STAT3 genes. Gray boxes show conserved regions, broken boxes show unknown regions. The numbers signify exons. AciI restriction enzyme sites in upstream of the promoter (5′) and in the last half of exon 24 (3′) are shown by open arrowheads. The BglII site is shown by a closed arrowhead. (b) RT–PCR of human STAT3 from CHO/STAT. STAT3 transcripts (about 1 kb) were detected in HT1080, CHO and four CHO lines that contained human STAT3 (CHO/STAT) by RT–PCR using primers corresponding to exons 2 and 9. Digestion of the transcripts with BglII produced 0.5- and 0.4-kb fragments of human STAT3 transcripts from HT1080 and nondigested 1.0-kb fragments from endogenous STAT3 in CHO cells and all CHO/STAT cell lines. (c) DNA methylation analysis in the 5′ and 3′ regions of human STAT3. Genomic DNA from HT1080, ES/STAT, CHO/STAT, two ES/STAT(CHO) and four NIH/STAT(CHO) cell lines was digested with MscI and DraI, or AflII and MscI, with (+) or without (−) AciI, and subsequently hybridized with 5′ or 3′ probes. The 1.2-kb fragments from digestion with AciI indicate a completely methylated state. (d) Detection of human STAT3 transcripts. Human STAT3 transcripts were detected in HT1080, mouse ES, CHO, NIH/3T3, five ES/STAT(CHO) and six NIH/STAT(CHO) cell lines by RT–PCR using human-specific primers corresponding to exons 2 and 24. A 2.2-kb fragment of the human STAT3 transcript was detected in all five ES/STAT(CHO) cell lines, while transcript detection varied in the six NIH/STAT(CHO) cell lines.
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Figure 5: Transfer of human STAT3 chromatin architecture. (a) Comparison of human, mouse and Chinese hamster STAT3 genes. Gray boxes show conserved regions, broken boxes show unknown regions. The numbers signify exons. AciI restriction enzyme sites in upstream of the promoter (5′) and in the last half of exon 24 (3′) are shown by open arrowheads. The BglII site is shown by a closed arrowhead. (b) RT–PCR of human STAT3 from CHO/STAT. STAT3 transcripts (about 1 kb) were detected in HT1080, CHO and four CHO lines that contained human STAT3 (CHO/STAT) by RT–PCR using primers corresponding to exons 2 and 9. Digestion of the transcripts with BglII produced 0.5- and 0.4-kb fragments of human STAT3 transcripts from HT1080 and nondigested 1.0-kb fragments from endogenous STAT3 in CHO cells and all CHO/STAT cell lines. (c) DNA methylation analysis in the 5′ and 3′ regions of human STAT3. Genomic DNA from HT1080, ES/STAT, CHO/STAT, two ES/STAT(CHO) and four NIH/STAT(CHO) cell lines was digested with MscI and DraI, or AflII and MscI, with (+) or without (−) AciI, and subsequently hybridized with 5′ or 3′ probes. The 1.2-kb fragments from digestion with AciI indicate a completely methylated state. (d) Detection of human STAT3 transcripts. Human STAT3 transcripts were detected in HT1080, mouse ES, CHO, NIH/3T3, five ES/STAT(CHO) and six NIH/STAT(CHO) cell lines by RT–PCR using human-specific primers corresponding to exons 2 and 24. A 2.2-kb fragment of the human STAT3 transcript was detected in all five ES/STAT(CHO) cell lines, while transcript detection varied in the six NIH/STAT(CHO) cell lines.

Mentions: In order to examine the property of the de novo constructed gene locus, human STAT3 DNA was inserted into the 25-4 vector in CHO cells (CHO/STAT), which are often used for protein production. While the sequence of the Chinese hamster endogenous STAT3 gene was unknown, RT–PCR using primers for exons 2 and 9 of human STAT3, which are highly conserved between humans and mice, produced 0.9-kb fragments from both human and CHO cells (Figure 5a, b). Restriction analysis with BglII, a human STAT3-specific restriction site, indicated that human STAT3 was not expressed in any CHO/STAT cell line, despite active expression of hamster STAT3 (Figure 5a, b).Figure 5.


Manipulating transgenes using a chromosome vector.

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

Transfer of human STAT3 chromatin architecture. (a) Comparison of human, mouse and Chinese hamster STAT3 genes. Gray boxes show conserved regions, broken boxes show unknown regions. The numbers signify exons. AciI restriction enzyme sites in upstream of the promoter (5′) and in the last half of exon 24 (3′) are shown by open arrowheads. The BglII site is shown by a closed arrowhead. (b) RT–PCR of human STAT3 from CHO/STAT. STAT3 transcripts (about 1 kb) were detected in HT1080, CHO and four CHO lines that contained human STAT3 (CHO/STAT) by RT–PCR using primers corresponding to exons 2 and 9. Digestion of the transcripts with BglII produced 0.5- and 0.4-kb fragments of human STAT3 transcripts from HT1080 and nondigested 1.0-kb fragments from endogenous STAT3 in CHO cells and all CHO/STAT cell lines. (c) DNA methylation analysis in the 5′ and 3′ regions of human STAT3. Genomic DNA from HT1080, ES/STAT, CHO/STAT, two ES/STAT(CHO) and four NIH/STAT(CHO) cell lines was digested with MscI and DraI, or AflII and MscI, with (+) or without (−) AciI, and subsequently hybridized with 5′ or 3′ probes. The 1.2-kb fragments from digestion with AciI indicate a completely methylated state. (d) Detection of human STAT3 transcripts. Human STAT3 transcripts were detected in HT1080, mouse ES, CHO, NIH/3T3, five ES/STAT(CHO) and six NIH/STAT(CHO) cell lines by RT–PCR using human-specific primers corresponding to exons 2 and 24. A 2.2-kb fragment of the human STAT3 transcript was detected in all five ES/STAT(CHO) cell lines, while transcript detection varied in the six NIH/STAT(CHO) cell lines.
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Figure 5: Transfer of human STAT3 chromatin architecture. (a) Comparison of human, mouse and Chinese hamster STAT3 genes. Gray boxes show conserved regions, broken boxes show unknown regions. The numbers signify exons. AciI restriction enzyme sites in upstream of the promoter (5′) and in the last half of exon 24 (3′) are shown by open arrowheads. The BglII site is shown by a closed arrowhead. (b) RT–PCR of human STAT3 from CHO/STAT. STAT3 transcripts (about 1 kb) were detected in HT1080, CHO and four CHO lines that contained human STAT3 (CHO/STAT) by RT–PCR using primers corresponding to exons 2 and 9. Digestion of the transcripts with BglII produced 0.5- and 0.4-kb fragments of human STAT3 transcripts from HT1080 and nondigested 1.0-kb fragments from endogenous STAT3 in CHO cells and all CHO/STAT cell lines. (c) DNA methylation analysis in the 5′ and 3′ regions of human STAT3. Genomic DNA from HT1080, ES/STAT, CHO/STAT, two ES/STAT(CHO) and four NIH/STAT(CHO) cell lines was digested with MscI and DraI, or AflII and MscI, with (+) or without (−) AciI, and subsequently hybridized with 5′ or 3′ probes. The 1.2-kb fragments from digestion with AciI indicate a completely methylated state. (d) Detection of human STAT3 transcripts. Human STAT3 transcripts were detected in HT1080, mouse ES, CHO, NIH/3T3, five ES/STAT(CHO) and six NIH/STAT(CHO) cell lines by RT–PCR using human-specific primers corresponding to exons 2 and 24. A 2.2-kb fragment of the human STAT3 transcript was detected in all five ES/STAT(CHO) cell lines, while transcript detection varied in the six NIH/STAT(CHO) cell lines.
Mentions: In order to examine the property of the de novo constructed gene locus, human STAT3 DNA was inserted into the 25-4 vector in CHO cells (CHO/STAT), which are often used for protein production. While the sequence of the Chinese hamster endogenous STAT3 gene was unknown, RT–PCR using primers for exons 2 and 9 of human STAT3, which are highly conserved between humans and mice, produced 0.9-kb fragments from both human and CHO cells (Figure 5a, b). Restriction analysis with BglII, a human STAT3-specific restriction site, indicated that human STAT3 was not expressed in any CHO/STAT cell line, despite active expression of hamster STAT3 (Figure 5a, b).Figure 5.

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