Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci
<p>Abstract</p> <p>Background</p> <p>Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the ge...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2005-07-01
|
| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/5/21 |
| _version_ | 1818565774072086528 |
|---|---|
| author | Stromberg Gregory Compitello George Basu Joydeep Willard Huntington F Van Bokkelen Gil |
| author_facet | Stromberg Gregory Compitello George Basu Joydeep Willard Huntington F Van Bokkelen Gil |
| author_sort | Stromberg Gregory |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of <it>de novo </it>HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into <it>de novo </it>HACs.</p> <p>Results</p> <p>We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs) for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple <it>de novo </it>HACs containing large (100–200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and ß-globin. We report significant differences in the ability of different genomic loci to support <it>de novo </it>HAC formation, suggesting possible effects of <it>cis</it>-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection.</p> <p>Conclusion</p> <p>Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate <it>de novo </it>HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors.</p> |
| first_indexed | 2024-12-14T01:45:19Z |
| format | Article |
| id | doaj.art-20e266209aa042c2a0b880c5530b65d7 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-14T01:45:19Z |
| publishDate | 2005-07-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-20e266209aa042c2a0b880c5530b65d72022-12-21T23:21:35ZengBMCBMC Biotechnology1472-67502005-07-01512110.1186/1472-6750-5-21Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic lociStromberg GregoryCompitello GeorgeBasu JoydeepWillard Huntington FVan Bokkelen Gil<p>Abstract</p> <p>Background</p> <p>Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of <it>de novo </it>HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into <it>de novo </it>HACs.</p> <p>Results</p> <p>We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs) for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple <it>de novo </it>HACs containing large (100–200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and ß-globin. We report significant differences in the ability of different genomic loci to support <it>de novo </it>HAC formation, suggesting possible effects of <it>cis</it>-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection.</p> <p>Conclusion</p> <p>Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate <it>de novo </it>HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors.</p>http://www.biomedcentral.com/1472-6750/5/21 |
| spellingShingle | Stromberg Gregory Compitello George Basu Joydeep Willard Huntington F Van Bokkelen Gil Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci BMC Biotechnology |
| title | Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci |
| title_full | Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci |
| title_fullStr | Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci |
| title_full_unstemmed | Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci |
| title_short | Efficient assembly of <it>de novo </it>human artificial chromosomes from large genomic loci |
| title_sort | efficient assembly of it de novo it human artificial chromosomes from large genomic loci |
| url | http://www.biomedcentral.com/1472-6750/5/21 |
| work_keys_str_mv | AT stromberggregory efficientassemblyofitdenovoithumanartificialchromosomesfromlargegenomicloci AT compitellogeorge efficientassemblyofitdenovoithumanartificialchromosomesfromlargegenomicloci AT basujoydeep efficientassemblyofitdenovoithumanartificialchromosomesfromlargegenomicloci AT willardhuntingtonf efficientassemblyofitdenovoithumanartificialchromosomesfromlargegenomicloci AT vanbokkelengil efficientassemblyofitdenovoithumanartificialchromosomesfromlargegenomicloci |