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A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

Yamaguchi S, Kazuki Y, Nakayama Y, Nanba E, Oshimura M, Ohbayashi T - PLoS ONE (2011)

Bottom Line: The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis.The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities.The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University, Yonago, Japan.

ABSTRACT
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

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Strategy for construction of the multi-integrase HAC vector.(A) Construction of entry clones (pENTR) from gateway attB-flanked PCR products and donor vectors (pDONR). (B) Construction of the multi-integrase platform plasmid from four entry clones and a destination vector (pDEST). (C) Construction of the HAC vector containing the multi-integrase platform using Cre/loxP system.
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pone-0017267-g001: Strategy for construction of the multi-integrase HAC vector.(A) Construction of entry clones (pENTR) from gateway attB-flanked PCR products and donor vectors (pDONR). (B) Construction of the multi-integrase platform plasmid from four entry clones and a destination vector (pDEST). (C) Construction of the HAC vector containing the multi-integrase platform using Cre/loxP system.

Mentions: The multi-integrase platform had four attP phage integrase recombination sites and an FRT site. The platform plasmid was constructed in E. coli using the Multiple Gateway system, which is useful in high-throughput construction of plasmids carrying multiple DNA sequences [44], [45]. The polymerase chain reaction (PCR) fragments for FRT-PGK-ΦC31 attP, PGK-R4 attP, PGK-TP901-1 attP and PGK-Bxb1 attP contained the appropriate gateway attB sites. The four fragments were recombined into four different donor vectors (pDONR™221 P1-P5r, pDONR221 P5-P4, pDONR221 P4r-P3r, pDONR221 P3-P2) to create four entry clones (pENTR L1-FRT-PGK-ΦC31-R5, pENTR L5-PGK-R4-L4, pENTR R4-PGK-TP901-1-R3, pENTR L3-PGK-Bxb1-L2) (Fig. 1A). DNA sequencing showed that 95.0% of kanamycin-resistant E. coli transformant colonies were correctly targeted. The four pENTR vectors and a pDEST vector were mixed in the presence of LR clonase enzyme to generate the multi-integrase platform plasmid (Fig. 1B). Using DNA sequencing, 40.7% of ampicillin-resistant E. coli transformant colonies were correctly targeted. The multi-integrase platform plasmid carried a loxP sequence and the 3′ hypoxanthine phosphoribosyl transferase (HPRT) sequence. The HAC vector used in this study was 21HAC1 containing the 5′ HPRT-loxP site [39]. The HPRT gene expressed in the HAC vector conferred HAT-resistance after site-specific recombination with the Cre/loxP system. The platform plasmid and Cre expression vector were co-transfected into CHO (hprt−/−) cells carrying the HAC vector (Fig. 1C). PCR analyses using the primers (Trans L1/R1, loxP4548/hyg696 and ΦC31 F1/Bxb1 R3) showed that 50.0% HAT-resistant transfectants were correctly targeted. This ratio of circular plasmid insertion into the 5′ HPRT-loxP site on the HAC vector by HPRT gene reconstitution was similar to that observed previously (33.3%) [39]. This HAC vector was designated as the multi-integrase HAC (MI-HAC) vector. Using fluorescence in situ hybridization (FISH), the digoxigenin-labeled human COT1 DNA probe localized to the MI-HAC vector and the HAC vector was present as an independent minichromosome without integration into the host genome in all six randomly selected clones (data not shown). These results indicated that a multi-integrase platform can be cloned into the defined locus on the HAC vector by the Cre/loxP system.


A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

Yamaguchi S, Kazuki Y, Nakayama Y, Nanba E, Oshimura M, Ohbayashi T - PLoS ONE (2011)

Strategy for construction of the multi-integrase HAC vector.(A) Construction of entry clones (pENTR) from gateway attB-flanked PCR products and donor vectors (pDONR). (B) Construction of the multi-integrase platform plasmid from four entry clones and a destination vector (pDEST). (C) Construction of the HAC vector containing the multi-integrase platform using Cre/loxP system.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044732&req=5

pone-0017267-g001: Strategy for construction of the multi-integrase HAC vector.(A) Construction of entry clones (pENTR) from gateway attB-flanked PCR products and donor vectors (pDONR). (B) Construction of the multi-integrase platform plasmid from four entry clones and a destination vector (pDEST). (C) Construction of the HAC vector containing the multi-integrase platform using Cre/loxP system.
Mentions: The multi-integrase platform had four attP phage integrase recombination sites and an FRT site. The platform plasmid was constructed in E. coli using the Multiple Gateway system, which is useful in high-throughput construction of plasmids carrying multiple DNA sequences [44], [45]. The polymerase chain reaction (PCR) fragments for FRT-PGK-ΦC31 attP, PGK-R4 attP, PGK-TP901-1 attP and PGK-Bxb1 attP contained the appropriate gateway attB sites. The four fragments were recombined into four different donor vectors (pDONR™221 P1-P5r, pDONR221 P5-P4, pDONR221 P4r-P3r, pDONR221 P3-P2) to create four entry clones (pENTR L1-FRT-PGK-ΦC31-R5, pENTR L5-PGK-R4-L4, pENTR R4-PGK-TP901-1-R3, pENTR L3-PGK-Bxb1-L2) (Fig. 1A). DNA sequencing showed that 95.0% of kanamycin-resistant E. coli transformant colonies were correctly targeted. The four pENTR vectors and a pDEST vector were mixed in the presence of LR clonase enzyme to generate the multi-integrase platform plasmid (Fig. 1B). Using DNA sequencing, 40.7% of ampicillin-resistant E. coli transformant colonies were correctly targeted. The multi-integrase platform plasmid carried a loxP sequence and the 3′ hypoxanthine phosphoribosyl transferase (HPRT) sequence. The HAC vector used in this study was 21HAC1 containing the 5′ HPRT-loxP site [39]. The HPRT gene expressed in the HAC vector conferred HAT-resistance after site-specific recombination with the Cre/loxP system. The platform plasmid and Cre expression vector were co-transfected into CHO (hprt−/−) cells carrying the HAC vector (Fig. 1C). PCR analyses using the primers (Trans L1/R1, loxP4548/hyg696 and ΦC31 F1/Bxb1 R3) showed that 50.0% HAT-resistant transfectants were correctly targeted. This ratio of circular plasmid insertion into the 5′ HPRT-loxP site on the HAC vector by HPRT gene reconstitution was similar to that observed previously (33.3%) [39]. This HAC vector was designated as the multi-integrase HAC (MI-HAC) vector. Using fluorescence in situ hybridization (FISH), the digoxigenin-labeled human COT1 DNA probe localized to the MI-HAC vector and the HAC vector was present as an independent minichromosome without integration into the host genome in all six randomly selected clones (data not shown). These results indicated that a multi-integrase platform can be cloned into the defined locus on the HAC vector by the Cre/loxP system.

Bottom Line: The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis.The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities.The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University, Yonago, Japan.

ABSTRACT
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

Show MeSH