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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

Cloning vectors.A. A linear map of pCMVlac-dirTopo-AU1/P2A-αGFP (4.85 and 4.89 kbp in length). A PCR-amplified cDNA is inserted at the cloning site via Vaccinia topoisomerase I-mediated recombination. A cytomegalovirus promoter (PCMV) and a modified E.coli lactose promoter/operator (lacPOm) are located upstream of the cloning site, which drive gene expression in mammalian cells as well as in E.coli. The cloning site is followed by the AU1 epitope or the P2A sequence (AU1/P2A), lacZα (encoding a peptide of E. coli β-galactosidase Ile4-Arg47, used as a linker) and GFP (encoding a green fluorescence protein, used as an expression marker). A SV40 polyadenylation signal (pA) is located downstream of GFP. The plasmid carries a kanamycin/neomycin resistance gene (KmR) under a SV40 early promoter and a KmR promoter, followed by a herpes simplex virus thymidine kinase polyadenylation signal (pA). The vector contains replication origins of f1 and pUC. Some of the unique restriction sites are shown. The numbers indicate the position of nucleotides in kbp. B. The vector sequence between NheI and XbaI sites. The modified lacPO (between NheI and BspEI sites) contains two point mutations (in red letters) to eliminate ATGs and also to reduce the promoter activity. The cloning site (between BspEI and BamHI sites, its complementary sequence is also shown) contains two Nt.BspQI recognition sites, two Vaccinia topoisomerase I (Topo) recognition sites, and a unique EcoRV recognition site. Upon cloning, the fragment between two Topo recognition sites (in red letters) is replaced with a PCR-amplified insert DNA. The AU1 epitope or P2A sequence (between BamHI and SalI sites) is located right after the cloning site (in blue boxes). An out-of-frame ATG is created just before the sequences (in red italics). Capital letters above the nucleotide sequence represent single-letter amino acid codes (above the 2nd position of each codon). A slash in the P2A peptide sequence indicates the self-cleaving site. The modified lacZα (between SalI and EcoRI sites) contains silent mutations to eliminate several restriction sites and to adjust codon usages. GFP (between EcoRI and XhoI sites, encoding EGFP from Clontech) lacks its initiation codon. Asterisks indicate termination codons.
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pone.0139349.g001: Cloning vectors.A. A linear map of pCMVlac-dirTopo-AU1/P2A-αGFP (4.85 and 4.89 kbp in length). A PCR-amplified cDNA is inserted at the cloning site via Vaccinia topoisomerase I-mediated recombination. A cytomegalovirus promoter (PCMV) and a modified E.coli lactose promoter/operator (lacPOm) are located upstream of the cloning site, which drive gene expression in mammalian cells as well as in E.coli. The cloning site is followed by the AU1 epitope or the P2A sequence (AU1/P2A), lacZα (encoding a peptide of E. coli β-galactosidase Ile4-Arg47, used as a linker) and GFP (encoding a green fluorescence protein, used as an expression marker). A SV40 polyadenylation signal (pA) is located downstream of GFP. The plasmid carries a kanamycin/neomycin resistance gene (KmR) under a SV40 early promoter and a KmR promoter, followed by a herpes simplex virus thymidine kinase polyadenylation signal (pA). The vector contains replication origins of f1 and pUC. Some of the unique restriction sites are shown. The numbers indicate the position of nucleotides in kbp. B. The vector sequence between NheI and XbaI sites. The modified lacPO (between NheI and BspEI sites) contains two point mutations (in red letters) to eliminate ATGs and also to reduce the promoter activity. The cloning site (between BspEI and BamHI sites, its complementary sequence is also shown) contains two Nt.BspQI recognition sites, two Vaccinia topoisomerase I (Topo) recognition sites, and a unique EcoRV recognition site. Upon cloning, the fragment between two Topo recognition sites (in red letters) is replaced with a PCR-amplified insert DNA. The AU1 epitope or P2A sequence (between BamHI and SalI sites) is located right after the cloning site (in blue boxes). An out-of-frame ATG is created just before the sequences (in red italics). Capital letters above the nucleotide sequence represent single-letter amino acid codes (above the 2nd position of each codon). A slash in the P2A peptide sequence indicates the self-cleaving site. The modified lacZα (between SalI and EcoRI sites) contains silent mutations to eliminate several restriction sites and to adjust codon usages. GFP (between EcoRI and XhoI sites, encoding EGFP from Clontech) lacks its initiation codon. Asterisks indicate termination codons.

Mentions: To create an efficient and convenient cloning vector suitable for expression studies in mammalian cells, I designed a plasmid, pCMVlac-dirTopo-AU1-αGFP (Fig 1). In attempts to enhance cloning efficiency, positive blue white selection was incorporated into Vaccinia topoisomerase I–mediated cloning. A modified E.coli lac promoter/operator (lacPO, 57 bp in length) was placed just downstream of a CMV enhancer/promoter (Pcmv, 590 bp in length). Two point mutations were introduced to lacPO to reduce its promoter activity for limited expression in E.coli, and to eliminate ATGs so as not to attenuate eukaryotic translation. Downstream of the cloning site, an AU1 epitope, lacZα, and GFP were inserted in-frame. LacZα served as a linker between the cloned cDNA and GFP. To block translation of lacZα-GFP under the modified lacPO, in-frame ATGs were removed and two in-frame stop codons (within the cloning site) and an out-of-frame ATG (just before the AU1 sequence) were placed. Therefore, only when a cDNA fragment is properly inserted into the vector, the cloned gene is expressed as a GFP fusion protein with a LacZα linker peptide. Its expression in E.coli strains harboring lacZΔM15 (e.g. DH5α) will lead to formation of blue colonies on X-gal plates via α-complementation of β-galactosidase [17].


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)

Cloning vectors.A. A linear map of pCMVlac-dirTopo-AU1/P2A-αGFP (4.85 and 4.89 kbp in length). A PCR-amplified cDNA is inserted at the cloning site via Vaccinia topoisomerase I-mediated recombination. A cytomegalovirus promoter (PCMV) and a modified E.coli lactose promoter/operator (lacPOm) are located upstream of the cloning site, which drive gene expression in mammalian cells as well as in E.coli. The cloning site is followed by the AU1 epitope or the P2A sequence (AU1/P2A), lacZα (encoding a peptide of E. coli β-galactosidase Ile4-Arg47, used as a linker) and GFP (encoding a green fluorescence protein, used as an expression marker). A SV40 polyadenylation signal (pA) is located downstream of GFP. The plasmid carries a kanamycin/neomycin resistance gene (KmR) under a SV40 early promoter and a KmR promoter, followed by a herpes simplex virus thymidine kinase polyadenylation signal (pA). The vector contains replication origins of f1 and pUC. Some of the unique restriction sites are shown. The numbers indicate the position of nucleotides in kbp. B. The vector sequence between NheI and XbaI sites. The modified lacPO (between NheI and BspEI sites) contains two point mutations (in red letters) to eliminate ATGs and also to reduce the promoter activity. The cloning site (between BspEI and BamHI sites, its complementary sequence is also shown) contains two Nt.BspQI recognition sites, two Vaccinia topoisomerase I (Topo) recognition sites, and a unique EcoRV recognition site. Upon cloning, the fragment between two Topo recognition sites (in red letters) is replaced with a PCR-amplified insert DNA. The AU1 epitope or P2A sequence (between BamHI and SalI sites) is located right after the cloning site (in blue boxes). An out-of-frame ATG is created just before the sequences (in red italics). Capital letters above the nucleotide sequence represent single-letter amino acid codes (above the 2nd position of each codon). A slash in the P2A peptide sequence indicates the self-cleaving site. The modified lacZα (between SalI and EcoRI sites) contains silent mutations to eliminate several restriction sites and to adjust codon usages. GFP (between EcoRI and XhoI sites, encoding EGFP from Clontech) lacks its initiation codon. Asterisks indicate termination codons.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139349.g001: Cloning vectors.A. A linear map of pCMVlac-dirTopo-AU1/P2A-αGFP (4.85 and 4.89 kbp in length). A PCR-amplified cDNA is inserted at the cloning site via Vaccinia topoisomerase I-mediated recombination. A cytomegalovirus promoter (PCMV) and a modified E.coli lactose promoter/operator (lacPOm) are located upstream of the cloning site, which drive gene expression in mammalian cells as well as in E.coli. The cloning site is followed by the AU1 epitope or the P2A sequence (AU1/P2A), lacZα (encoding a peptide of E. coli β-galactosidase Ile4-Arg47, used as a linker) and GFP (encoding a green fluorescence protein, used as an expression marker). A SV40 polyadenylation signal (pA) is located downstream of GFP. The plasmid carries a kanamycin/neomycin resistance gene (KmR) under a SV40 early promoter and a KmR promoter, followed by a herpes simplex virus thymidine kinase polyadenylation signal (pA). The vector contains replication origins of f1 and pUC. Some of the unique restriction sites are shown. The numbers indicate the position of nucleotides in kbp. B. The vector sequence between NheI and XbaI sites. The modified lacPO (between NheI and BspEI sites) contains two point mutations (in red letters) to eliminate ATGs and also to reduce the promoter activity. The cloning site (between BspEI and BamHI sites, its complementary sequence is also shown) contains two Nt.BspQI recognition sites, two Vaccinia topoisomerase I (Topo) recognition sites, and a unique EcoRV recognition site. Upon cloning, the fragment between two Topo recognition sites (in red letters) is replaced with a PCR-amplified insert DNA. The AU1 epitope or P2A sequence (between BamHI and SalI sites) is located right after the cloning site (in blue boxes). An out-of-frame ATG is created just before the sequences (in red italics). Capital letters above the nucleotide sequence represent single-letter amino acid codes (above the 2nd position of each codon). A slash in the P2A peptide sequence indicates the self-cleaving site. The modified lacZα (between SalI and EcoRI sites) contains silent mutations to eliminate several restriction sites and to adjust codon usages. GFP (between EcoRI and XhoI sites, encoding EGFP from Clontech) lacks its initiation codon. Asterisks indicate termination codons.
Mentions: To create an efficient and convenient cloning vector suitable for expression studies in mammalian cells, I designed a plasmid, pCMVlac-dirTopo-AU1-αGFP (Fig 1). In attempts to enhance cloning efficiency, positive blue white selection was incorporated into Vaccinia topoisomerase I–mediated cloning. A modified E.coli lac promoter/operator (lacPO, 57 bp in length) was placed just downstream of a CMV enhancer/promoter (Pcmv, 590 bp in length). Two point mutations were introduced to lacPO to reduce its promoter activity for limited expression in E.coli, and to eliminate ATGs so as not to attenuate eukaryotic translation. Downstream of the cloning site, an AU1 epitope, lacZα, and GFP were inserted in-frame. LacZα served as a linker between the cloned cDNA and GFP. To block translation of lacZα-GFP under the modified lacPO, in-frame ATGs were removed and two in-frame stop codons (within the cloning site) and an out-of-frame ATG (just before the AU1 sequence) were placed. Therefore, only when a cDNA fragment is properly inserted into the vector, the cloned gene is expressed as a GFP fusion protein with a LacZα linker peptide. Its expression in E.coli strains harboring lacZΔM15 (e.g. DH5α) will lead to formation of blue colonies on X-gal plates via α-complementation of β-galactosidase [17].

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