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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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Comparison of the efficiency in generating transgenic cells.(A) Schematic diagram of the insertion of the plasmid containing the CMV promoter and the EGFP gene in the multi-integrase HAC vector or host chromosome. Under both conditions, cells were incubated with the reagent/DNA mixture for 24 h and selected with G418 until drug-resistant cell pools were obtained. (B) Flow cytometry analysis of the fluorescent profile of transfected CHO cells on day 12 following selection by G418 after transfection. The bars indicate the GFP-expressing cell population. (C) Expression of the inserted EGFP gene was tracked in cells by fluorescence microscopy. Typical images (bright field, upper panels; fluorescence, lower panels) of cells obtained by the multi-integrase HAC vector with integrases and random integration are shown. Scale bar, 100 µm.
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pone-0017267-g003: Comparison of the efficiency in generating transgenic cells.(A) Schematic diagram of the insertion of the plasmid containing the CMV promoter and the EGFP gene in the multi-integrase HAC vector or host chromosome. Under both conditions, cells were incubated with the reagent/DNA mixture for 24 h and selected with G418 until drug-resistant cell pools were obtained. (B) Flow cytometry analysis of the fluorescent profile of transfected CHO cells on day 12 following selection by G418 after transfection. The bars indicate the GFP-expressing cell population. (C) Expression of the inserted EGFP gene was tracked in cells by fluorescence microscopy. Typical images (bright field, upper panels; fluorescence, lower panels) of cells obtained by the multi-integrase HAC vector with integrases and random integration are shown. Scale bar, 100 µm.

Mentions: Based on the likely activity of phage integrases in mediating recombination events in CHO cells, we tested whether the MI-HAC vector with integrases was able to produce cells expressing the gene of interest more efficiently than the conventional random integration method. The transfection experiments were carried out with pNeo-attB EGFP or pEGFP N1, which both contained the strong CMV enhancer coupled to the enhanced green fluorescent protein (EGFP) gene (Fig. 3A). Before transfection, the pEGFP N1 was linearized at the AseI site, upstream from the CMV promoter. The host cells used in the transfection experiments were CHO cells carrying the MI-HAC vector. Each transfection was subjected to G418 selection, and colony numbers were assessed. In one such experiment, approximately 5–17-fold more colonies under G418 selective pressure for 12 days were observed when the linearized pEGFP N1 was transfected into CHO cells (random integration method), compared with pNeo-attB EGFP with the correct integrase expression plasmid (MI-HAC method). Following transfection after 12 days in G418 selection medium, the transfected cells were analyzed by flow cytometry to examine the proportion of GFP expression CHO colonies. As a result, the GFP-positive cells obtained by the MI-HAC method with ΦC31 integrase comprised 89.9% of the total cell population (Fig. 3B, Table 1). The negative control employed was CHO cells without a GFP expression cassette. Similar experiments using R4, TP901-1 and Bxb1 integrases demonstrated the proportion of GFP-positive cells to be 71.4, 75.7 and 75.0%, respectively (Fig. 3B, Table 1). Although GFP-positive cells obtained by the random integration method comprised 59.6%, there was not a great difference in the proportion of GFP-positive cells by the MI-HAC vector or the random integration methods (Table 1). However, we observed a wide distribution of GFP expression in the pool of cells obtained following application of the random integration method (Fig. 3B). In addition, we observed the expression of GFP using fluorescence microscopy in CHO colonies. As a result, in colonies obtained by the MI-HAC method, we observed homogenous GFP expression (nearly every cell expressed GFP in the clonal population) in many colonies (Fig. 3C, Table 1). In contrast, we observed only 10.0% of colonies with homogenous GFP expression after the random integration method (Table 1). Nearly 90.0% of colonies consisted of heterogenous GFP-expressing cells (a mixture of very little or no GFP-expressing cells in the clonal population) following random integration (Fig. 3C).


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)

Comparison of the efficiency in generating transgenic cells.(A) Schematic diagram of the insertion of the plasmid containing the CMV promoter and the EGFP gene in the multi-integrase HAC vector or host chromosome. Under both conditions, cells were incubated with the reagent/DNA mixture for 24 h and selected with G418 until drug-resistant cell pools were obtained. (B) Flow cytometry analysis of the fluorescent profile of transfected CHO cells on day 12 following selection by G418 after transfection. The bars indicate the GFP-expressing cell population. (C) Expression of the inserted EGFP gene was tracked in cells by fluorescence microscopy. Typical images (bright field, upper panels; fluorescence, lower panels) of cells obtained by the multi-integrase HAC vector with integrases and random integration are shown. Scale bar, 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017267-g003: Comparison of the efficiency in generating transgenic cells.(A) Schematic diagram of the insertion of the plasmid containing the CMV promoter and the EGFP gene in the multi-integrase HAC vector or host chromosome. Under both conditions, cells were incubated with the reagent/DNA mixture for 24 h and selected with G418 until drug-resistant cell pools were obtained. (B) Flow cytometry analysis of the fluorescent profile of transfected CHO cells on day 12 following selection by G418 after transfection. The bars indicate the GFP-expressing cell population. (C) Expression of the inserted EGFP gene was tracked in cells by fluorescence microscopy. Typical images (bright field, upper panels; fluorescence, lower panels) of cells obtained by the multi-integrase HAC vector with integrases and random integration are shown. Scale bar, 100 µm.
Mentions: Based on the likely activity of phage integrases in mediating recombination events in CHO cells, we tested whether the MI-HAC vector with integrases was able to produce cells expressing the gene of interest more efficiently than the conventional random integration method. The transfection experiments were carried out with pNeo-attB EGFP or pEGFP N1, which both contained the strong CMV enhancer coupled to the enhanced green fluorescent protein (EGFP) gene (Fig. 3A). Before transfection, the pEGFP N1 was linearized at the AseI site, upstream from the CMV promoter. The host cells used in the transfection experiments were CHO cells carrying the MI-HAC vector. Each transfection was subjected to G418 selection, and colony numbers were assessed. In one such experiment, approximately 5–17-fold more colonies under G418 selective pressure for 12 days were observed when the linearized pEGFP N1 was transfected into CHO cells (random integration method), compared with pNeo-attB EGFP with the correct integrase expression plasmid (MI-HAC method). Following transfection after 12 days in G418 selection medium, the transfected cells were analyzed by flow cytometry to examine the proportion of GFP expression CHO colonies. As a result, the GFP-positive cells obtained by the MI-HAC method with ΦC31 integrase comprised 89.9% of the total cell population (Fig. 3B, Table 1). The negative control employed was CHO cells without a GFP expression cassette. Similar experiments using R4, TP901-1 and Bxb1 integrases demonstrated the proportion of GFP-positive cells to be 71.4, 75.7 and 75.0%, respectively (Fig. 3B, Table 1). Although GFP-positive cells obtained by the random integration method comprised 59.6%, there was not a great difference in the proportion of GFP-positive cells by the MI-HAC vector or the random integration methods (Table 1). However, we observed a wide distribution of GFP expression in the pool of cells obtained following application of the random integration method (Fig. 3B). In addition, we observed the expression of GFP using fluorescence microscopy in CHO colonies. As a result, in colonies obtained by the MI-HAC method, we observed homogenous GFP expression (nearly every cell expressed GFP in the clonal population) in many colonies (Fig. 3C, Table 1). In contrast, we observed only 10.0% of colonies with homogenous GFP expression after the random integration method (Table 1). Nearly 90.0% of colonies consisted of heterogenous GFP-expressing cells (a mixture of very little or no GFP-expressing cells in the clonal population) following random integration (Fig. 3C).

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