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A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line.

Tammur J, Sibul H, Ustav E, Ustav M, Metspalu A - BMC Mol. Biol. (2002)

Bottom Line: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles.The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation.In vivo experiments should reveal, whether 1-5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cell Biology, Tartu University, Estonian Biocentre, 23 Riia St, 51010 Tartu, Estonia. jtammur@ebc.ee

ABSTRACT

Background: The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7.

Results: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles.

Conclusion: Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1-5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

No MeSH data available.


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Figure 4a. Fluorescence in situ analysis of the p3.7LDL-transfected CHO-ldlA7 cells. The probe, generated from the p3.7LDL gives cross-hybridisation signals represented by yellow double dots on chromosomes stained with propidium iodide. This can be explained by integration of the vector sequences into the host cell genome The discrete yellow dots dispersed all over the chromosomes also correspond to plasmid-specific signals and suggest the presence of an episomal vector.Figure 4b. Fluorescence in situ analysis of the native CHO-ldlA7 cells (negative control).
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Figure 4: Figure 4a. Fluorescence in situ analysis of the p3.7LDL-transfected CHO-ldlA7 cells. The probe, generated from the p3.7LDL gives cross-hybridisation signals represented by yellow double dots on chromosomes stained with propidium iodide. This can be explained by integration of the vector sequences into the host cell genome The discrete yellow dots dispersed all over the chromosomes also correspond to plasmid-specific signals and suggest the presence of an episomal vector.Figure 4b. Fluorescence in situ analysis of the native CHO-ldlA7 cells (negative control).

Mentions: Integration of the p3.7LDL sequences was further confirmed by FISH (Fig. 4a). The sequence of the expression vector integrated into the genome gives cross-hybridisation signals represented by yellow double dots on the metaphase chromosomes. The speckled appearance of the vector signal indicates the episomal maintenance of the introduced vector. No such signals were observed in the FISH analysis of native CHO-ldlA7 cells (Fig. 4b). The possibility of integration of the BPV-1 based vectors in some cell lines has been also demonstrated by Ohe et al. [15]. However, the mechanism of integration has not been clearly defined yet. Next, we looked for presence of the LDLR protein in transfected receptor-deficient cells by immunofluorescence assay (Fig. 6). The protein-specific fluorescent signal was present in 1–5% of the examined cells, localising mainly in the Golgi complex area, where post-translational processing of the protein occurs; the signal was also observed to be evenly associated with the cell membrane (Fig. 6a) while native CHO-ldlA7 cells displayed only faint background fluorescent signal (Fig. 6b). To determine the functional properties of the transgene LDLR we studied its ability to internalise LDL particles. The DiI-labeled LDL particles were added into the culture medium of transfected and native CHO-ldlA7 cells. After three hours of incubation and fixation, the transfected cells displayed a strong signal inside the cell, attributable to the internalisation of the labelled particles (Fig. 7).


A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line.

Tammur J, Sibul H, Ustav E, Ustav M, Metspalu A - BMC Mol. Biol. (2002)

Figure 4a. Fluorescence in situ analysis of the p3.7LDL-transfected CHO-ldlA7 cells. The probe, generated from the p3.7LDL gives cross-hybridisation signals represented by yellow double dots on chromosomes stained with propidium iodide. This can be explained by integration of the vector sequences into the host cell genome The discrete yellow dots dispersed all over the chromosomes also correspond to plasmid-specific signals and suggest the presence of an episomal vector.Figure 4b. Fluorescence in situ analysis of the native CHO-ldlA7 cells (negative control).
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Related In: Results  -  Collection

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Figure 4: Figure 4a. Fluorescence in situ analysis of the p3.7LDL-transfected CHO-ldlA7 cells. The probe, generated from the p3.7LDL gives cross-hybridisation signals represented by yellow double dots on chromosomes stained with propidium iodide. This can be explained by integration of the vector sequences into the host cell genome The discrete yellow dots dispersed all over the chromosomes also correspond to plasmid-specific signals and suggest the presence of an episomal vector.Figure 4b. Fluorescence in situ analysis of the native CHO-ldlA7 cells (negative control).
Mentions: Integration of the p3.7LDL sequences was further confirmed by FISH (Fig. 4a). The sequence of the expression vector integrated into the genome gives cross-hybridisation signals represented by yellow double dots on the metaphase chromosomes. The speckled appearance of the vector signal indicates the episomal maintenance of the introduced vector. No such signals were observed in the FISH analysis of native CHO-ldlA7 cells (Fig. 4b). The possibility of integration of the BPV-1 based vectors in some cell lines has been also demonstrated by Ohe et al. [15]. However, the mechanism of integration has not been clearly defined yet. Next, we looked for presence of the LDLR protein in transfected receptor-deficient cells by immunofluorescence assay (Fig. 6). The protein-specific fluorescent signal was present in 1–5% of the examined cells, localising mainly in the Golgi complex area, where post-translational processing of the protein occurs; the signal was also observed to be evenly associated with the cell membrane (Fig. 6a) while native CHO-ldlA7 cells displayed only faint background fluorescent signal (Fig. 6b). To determine the functional properties of the transgene LDLR we studied its ability to internalise LDL particles. The DiI-labeled LDL particles were added into the culture medium of transfected and native CHO-ldlA7 cells. After three hours of incubation and fixation, the transfected cells displayed a strong signal inside the cell, attributable to the internalisation of the labelled particles (Fig. 7).

Bottom Line: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles.The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation.In vivo experiments should reveal, whether 1-5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cell Biology, Tartu University, Estonian Biocentre, 23 Riia St, 51010 Tartu, Estonia. jtammur@ebc.ee

ABSTRACT

Background: The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7.

Results: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles.

Conclusion: Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1-5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

No MeSH data available.


Related in: MedlinePlus