Construction of human artificial chromosome vectors by recombineering

Kotzamanis, George; Cheung, Wing Ming Francis; Abdulrazzak, Hassan; Pérez-Luz, Sara; Howe, Steven J.; Cooke, Howard J.; Huxley, Clare

doi:10.1016/j.gene.2005.01.017

Cited by 39 publications

(28 citation statements)

References 21 publications

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“…A number of in vivo site-specific recombination approaches to join large alpha satellite arrays with large genomic fragments have been reported [32,33], involving multi-step recombinogenic methodologies whose limitations are evidenced by the fact that few genomic loci have to date been reported to have been successfully incorporated into a HAC vector. In contrast, our transposon-based strategy for the construction of bimolecular BAC-based HAC vectors has facilitated the high-throughput creation of de novo HACs from multiple large genomic loci.…”

Section: Discussionmentioning

confidence: 99%

Efficient assembly of de novohuman artificial chromosomes from large genomic loci

et al. 2005

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BackgroundHuman 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 de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs.ResultsWe 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 de novo 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 de novo HAC formation, suggesting possible effects of cis-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.ConclusionTaken 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 de novo HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors.

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Section: Discussionmentioning

confidence: 99%

Efficient assembly of de novohuman artificial chromosomes from large genomic loci

et al. 2005

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“…FISH analysis was carried out on CMTCFOE clones approximately 5 weeks after their generation by bacterial invasion as described previously [41]. Human CFTR sequences were detected with a probe made of DNA from BAC CFTR1, 2, 3 labelled using the Biotin-Nick Translation Mix (Roche Burgess Hill, UK).…”

Section: Methodsmentioning

confidence: 99%

CFTR expression from a BAC carrying the complete human gene and associated regulatory elements

Kotzamanis

Abdulrazzak

Gifford-Garner

et al. 2009

J Cellular Molecular Medi

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The use of genomic DNA rather than cDNA or mini-gene constructs in gene therapy might be advantageous as these contain intronic and long-range control elements vital for accurate expression. For gene therapy of cystic fibrosis though, no bacterial artificial chromosome (BAC), containing the whole CFTR gene is available. We have used Red homologous recombination to add a to a previously described vector to construct a new BAC vector with a 250.3-kb insert containing the whole coding region of the CFTR gene along with 40.1 kb of DNA 5′ to the gene and 25 kb 3′ to the gene. This includes all the known control elements of the gene. We evaluated expression by RT-PCR in CMT-93 cells and showed that the gene is expressed both from integrated copies of the BAC and also from episomes carrying the oriP/EBNA-1 element. Sequencing of the human CFTR mRNA from one clone showed that the BAC is functional and can generate correctly spliced mRNA in the mouse background. The BAC described here is the only CFTR genomic construct available on a convenient vector that can be readily used for gene expression studies or in vivo studies to test its potential application in gene therapy for cystic fibrosis.

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“…The potential use of these vectors in gene therapy has been demonstrated by expression of large therapeutic genes from them (Grimes et al, 2001;Mejía & Larin, 2000). Further advance was noted when efficient methods for manipulating large sequences of repetitive nature, such as alphoid DNA, were developed and used to generate HACs, as will be discussed later on (Kotzamanis et al, 2005). Nevertheless, several issues need to be solved before any clinical application.…”

Section: Human Artificial Chromosomesmentioning

confidence: 99%

“…Particularly the Red recombination system has been used to introduce a 70-kb alphoid array into a BAC, carrying a 156-kb genomic insert containing the HPRT gene, by recombineering (Figure 2) and expression of the HPRT gene from generated HACs has been demonstrated (Kotzamanis et al, 2005). As shown in Figure 2, recombination was targeted to the chloramphenicol resistance gene which is present on all BACs, allowing for the addition of the 70-kb alphoid array into any BAC and subsequent formation of HACs in the appropriate cells (Kotzamanis et al, 2005). …”

Section: Addition Of Large Sequencesmentioning

confidence: 99%