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An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination.

Udo H - PLoS ONE (2015)

Bottom Line: However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter).When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates.This system provides an alternative method for cDNA cloning and expression in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Science, Kyushu University, Fukuoka, Japan.

ABSTRACT
One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

cDNA expression in mammalian cells.A. Fluorescence imaging of mammalian cells expressing Pax6. Mouse Pax6 (a transcription factor involved in neurogenesis) was cloned in pCMVlac-dirTopo-AU1-αGFP and pCMVlac-dirTopo-P2A-αGFP, and expressed in HEK293 cells. Vector controls and pEGFP-C2 (GFP control) were also included in this analysis. Expression and subcellular localization of the gene products were identified by GFP fluorescence (green). Cell nuclei were visualized with propidium iodide (PI, red), and Pax6 was immunostained (blue). Pax6 was exclusively localized in the nuclei. When Pax6 was expressed using the P2A vector, GFP fluorescence tended to diffusely distribute in the cytoplasm as a result of self-cleavage at P2A. Scale bar, 5 μm. B. Western blotting of transfected cells. Pax6 was expressed using the AU1 and P2A vectors in HEK293 cells. Whole cell, nucleus, and cytosol fractions were prepared and probed with an anti-Pax6 antibody (top panel) or anti-β-tubulin antibody (bottom, as an internal control). The vector controls did not have any Pax6 staining. GFP-fused Pax6 was detected at 81 kDa and localized exclusively in the nucleus. About one-half (53%) of Pax6-P2A-αGFP, but not Pax6-AU1-αGFP, was self-cleaved, which produced a 49 kDa band (cleaved Pax6) localizing in the nucleus.β-tubulin (50 kDa) was absent from the nucleus fraction. The numbers on the left indicate the size of pre-stained marker proteins in kDa.
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pone.0139349.g003: cDNA expression in mammalian cells.A. Fluorescence imaging of mammalian cells expressing Pax6. Mouse Pax6 (a transcription factor involved in neurogenesis) was cloned in pCMVlac-dirTopo-AU1-αGFP and pCMVlac-dirTopo-P2A-αGFP, and expressed in HEK293 cells. Vector controls and pEGFP-C2 (GFP control) were also included in this analysis. Expression and subcellular localization of the gene products were identified by GFP fluorescence (green). Cell nuclei were visualized with propidium iodide (PI, red), and Pax6 was immunostained (blue). Pax6 was exclusively localized in the nuclei. When Pax6 was expressed using the P2A vector, GFP fluorescence tended to diffusely distribute in the cytoplasm as a result of self-cleavage at P2A. Scale bar, 5 μm. B. Western blotting of transfected cells. Pax6 was expressed using the AU1 and P2A vectors in HEK293 cells. Whole cell, nucleus, and cytosol fractions were prepared and probed with an anti-Pax6 antibody (top panel) or anti-β-tubulin antibody (bottom, as an internal control). The vector controls did not have any Pax6 staining. GFP-fused Pax6 was detected at 81 kDa and localized exclusively in the nucleus. About one-half (53%) of Pax6-P2A-αGFP, but not Pax6-AU1-αGFP, was self-cleaved, which produced a 49 kDa band (cleaved Pax6) localizing in the nucleus.β-tubulin (50 kDa) was absent from the nucleus fraction. The numbers on the left indicate the size of pre-stained marker proteins in kDa.

Mentions: Recombinant plasmids can be directly used for expression studies in mammalian cells. Expression of the gene product can be easily monitored by GFP fluorescence. In this study, I also designed a plasmid containing a P2A sequence [9, 10] in place of AU1 (see Fig 1). With this vector, fusion proteins may be self-cleaved at the P2A site to release the fused LacZα-GFP. Fig 3A shows the expression of mouse Pax6 (a transcription factor involved in neuronal differentiation) in HEK293 cells. When Pax6 was expressed using the AU1 vector, both GFP fluorescence and Pax6 immunostaining were detected in the cell nuclei (indicated by PI staining). In contrast, when Pax6 was expressed using the P2A vector, GFP fluorescence tended to diffusely distribute throughout the cytoplasm, while Pax6 immunostaining was observed only in the nucleus. Expression of GFP alone visualized the whole cell without any Pax6 signals. Vector controls only showed PI staining in the nucleus under 30–40% transfection efficiency. The efficiency of self-cleavage by P2A was examined by Western blotting (Fig 3B). The result indicated that 55.2 ± 3.6% (n = 3 tests) of Pax6-P2A-αGFP (81 kDa) was self-cleaved, producing a 49 kDa band of Pax6. Both GFP-fused and cleaved Pax6 were detected in the nucleus fraction (β-tubulin, as an internal control, was absent in the nucleus fraction). It suggests that at least a part of LacZα-GFP was cleaved off by P2A. Although green fluorescence of GFP was generally weaker with the P2A vector, it still functioned as an expression marker for transfected cells. The P2A vector may be useful when the functionality of the fusion protein is concerned.


An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination.

Udo H - PLoS ONE (2015)

cDNA expression in mammalian cells.A. Fluorescence imaging of mammalian cells expressing Pax6. Mouse Pax6 (a transcription factor involved in neurogenesis) was cloned in pCMVlac-dirTopo-AU1-αGFP and pCMVlac-dirTopo-P2A-αGFP, and expressed in HEK293 cells. Vector controls and pEGFP-C2 (GFP control) were also included in this analysis. Expression and subcellular localization of the gene products were identified by GFP fluorescence (green). Cell nuclei were visualized with propidium iodide (PI, red), and Pax6 was immunostained (blue). Pax6 was exclusively localized in the nuclei. When Pax6 was expressed using the P2A vector, GFP fluorescence tended to diffusely distribute in the cytoplasm as a result of self-cleavage at P2A. Scale bar, 5 μm. B. Western blotting of transfected cells. Pax6 was expressed using the AU1 and P2A vectors in HEK293 cells. Whole cell, nucleus, and cytosol fractions were prepared and probed with an anti-Pax6 antibody (top panel) or anti-β-tubulin antibody (bottom, as an internal control). The vector controls did not have any Pax6 staining. GFP-fused Pax6 was detected at 81 kDa and localized exclusively in the nucleus. About one-half (53%) of Pax6-P2A-αGFP, but not Pax6-AU1-αGFP, was self-cleaved, which produced a 49 kDa band (cleaved Pax6) localizing in the nucleus.β-tubulin (50 kDa) was absent from the nucleus fraction. The numbers on the left indicate the size of pre-stained marker proteins in kDa.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4589362&req=5

pone.0139349.g003: cDNA expression in mammalian cells.A. Fluorescence imaging of mammalian cells expressing Pax6. Mouse Pax6 (a transcription factor involved in neurogenesis) was cloned in pCMVlac-dirTopo-AU1-αGFP and pCMVlac-dirTopo-P2A-αGFP, and expressed in HEK293 cells. Vector controls and pEGFP-C2 (GFP control) were also included in this analysis. Expression and subcellular localization of the gene products were identified by GFP fluorescence (green). Cell nuclei were visualized with propidium iodide (PI, red), and Pax6 was immunostained (blue). Pax6 was exclusively localized in the nuclei. When Pax6 was expressed using the P2A vector, GFP fluorescence tended to diffusely distribute in the cytoplasm as a result of self-cleavage at P2A. Scale bar, 5 μm. B. Western blotting of transfected cells. Pax6 was expressed using the AU1 and P2A vectors in HEK293 cells. Whole cell, nucleus, and cytosol fractions were prepared and probed with an anti-Pax6 antibody (top panel) or anti-β-tubulin antibody (bottom, as an internal control). The vector controls did not have any Pax6 staining. GFP-fused Pax6 was detected at 81 kDa and localized exclusively in the nucleus. About one-half (53%) of Pax6-P2A-αGFP, but not Pax6-AU1-αGFP, was self-cleaved, which produced a 49 kDa band (cleaved Pax6) localizing in the nucleus.β-tubulin (50 kDa) was absent from the nucleus fraction. The numbers on the left indicate the size of pre-stained marker proteins in kDa.
Mentions: Recombinant plasmids can be directly used for expression studies in mammalian cells. Expression of the gene product can be easily monitored by GFP fluorescence. In this study, I also designed a plasmid containing a P2A sequence [9, 10] in place of AU1 (see Fig 1). With this vector, fusion proteins may be self-cleaved at the P2A site to release the fused LacZα-GFP. Fig 3A shows the expression of mouse Pax6 (a transcription factor involved in neuronal differentiation) in HEK293 cells. When Pax6 was expressed using the AU1 vector, both GFP fluorescence and Pax6 immunostaining were detected in the cell nuclei (indicated by PI staining). In contrast, when Pax6 was expressed using the P2A vector, GFP fluorescence tended to diffusely distribute throughout the cytoplasm, while Pax6 immunostaining was observed only in the nucleus. Expression of GFP alone visualized the whole cell without any Pax6 signals. Vector controls only showed PI staining in the nucleus under 30–40% transfection efficiency. The efficiency of self-cleavage by P2A was examined by Western blotting (Fig 3B). The result indicated that 55.2 ± 3.6% (n = 3 tests) of Pax6-P2A-αGFP (81 kDa) was self-cleaved, producing a 49 kDa band of Pax6. Both GFP-fused and cleaved Pax6 were detected in the nucleus fraction (β-tubulin, as an internal control, was absent in the nucleus fraction). It suggests that at least a part of LacZα-GFP was cleaved off by P2A. Although green fluorescence of GFP was generally weaker with the P2A vector, it still functioned as an expression marker for transfected cells. The P2A vector may be useful when the functionality of the fusion protein is concerned.

Bottom Line: However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter).When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates.This system provides an alternative method for cDNA cloning and expression in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Science, Kyushu University, Fukuoka, Japan.

ABSTRACT
One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.

No MeSH data available.


Related in: MedlinePlus