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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

Simplified preparation of Vaccinia topoisomerase I-linked vector.A. Schematic diagram of vector preparation. The procedure consisted of successive enzymatic reactions with a single precipitation step. The vector DNA was first digested with Nt.BspQI (5’-GCTCTTCN↓-3’, black arrows indicate the nicking sites) for 1 hr at 50°C, heated at 80°C for 20 min, and linearized with EcoRV (5’-GAT↓ATC-3’) for 1 hr at 37°C. These treatments produced two 24-nucleotide fragments (in green letters) which remained bound to the vector DNA (Tm 75.5°C). Purified Vaccinia topoisomerase I (5’-CCCTT↓-3’, red arrows indicate the nicking sites) was then added and linked with the vector DNA for 5 min at 37°C. Finally, the enzyme-linked vector was precipitated with 10% polyethyleneglycol (PEG, Mw 8,000) at 14,000 rpm for 20 min at 4°C. The whole process was completed in 3 hr. B. Agarose gel electrophoresis of the vector DNA at each step of preparation. The uncut vector was comprised of super coiled (SC) and open circular (OC) conformations (lane 2). Digestion with Nt.BspQI yielded nicked plasmids, which migrated at the open circular position (lane 3). EcoRV digestion linearized the vector DNA, producing a single band at 4.85 kb (lane 4). Addition of Vaccinia topoisomerase I resulted in band shift of the vector DNA (lane 5). Some DNAs were hyper-linked (smeared appearance) or aggregated in the loading well. Precipitation with 10% PEG recovered the enzyme-linked vector, but most of the hyper-linked and aggregated DNAs were removed (lane 6). Marker, λ/HindIII (the numbers indicate the size of DNA fragments in kbp).
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pone.0139349.g004: Simplified preparation of Vaccinia topoisomerase I-linked vector.A. Schematic diagram of vector preparation. The procedure consisted of successive enzymatic reactions with a single precipitation step. The vector DNA was first digested with Nt.BspQI (5’-GCTCTTCN↓-3’, black arrows indicate the nicking sites) for 1 hr at 50°C, heated at 80°C for 20 min, and linearized with EcoRV (5’-GAT↓ATC-3’) for 1 hr at 37°C. These treatments produced two 24-nucleotide fragments (in green letters) which remained bound to the vector DNA (Tm 75.5°C). Purified Vaccinia topoisomerase I (5’-CCCTT↓-3’, red arrows indicate the nicking sites) was then added and linked with the vector DNA for 5 min at 37°C. Finally, the enzyme-linked vector was precipitated with 10% polyethyleneglycol (PEG, Mw 8,000) at 14,000 rpm for 20 min at 4°C. The whole process was completed in 3 hr. B. Agarose gel electrophoresis of the vector DNA at each step of preparation. The uncut vector was comprised of super coiled (SC) and open circular (OC) conformations (lane 2). Digestion with Nt.BspQI yielded nicked plasmids, which migrated at the open circular position (lane 3). EcoRV digestion linearized the vector DNA, producing a single band at 4.85 kb (lane 4). Addition of Vaccinia topoisomerase I resulted in band shift of the vector DNA (lane 5). Some DNAs were hyper-linked (smeared appearance) or aggregated in the loading well. Precipitation with 10% PEG recovered the enzyme-linked vector, but most of the hyper-linked and aggregated DNAs were removed (lane 6). Marker, λ/HindIII (the numbers indicate the size of DNA fragments in kbp).

Mentions: In the course of experiments, preparation of Vaccinia topoisomerase I-linked vector was streamlined. It consisted of successive enzymatic reactions with a single precipitation step, and the whole process was completed in 3 hr (Fig 4A). For preparation, the vector DNA was first digested with Nt.BspQI, linearized with EcoRV, and then linked with Vaccinia topoisomerase I followed by precipitation with 10% PEG. Fig 4B illustrates agarose gel electrophoresis of the vector DNA at each step of preparation. The uncut vector consisted of super-coiled and open circular forms. After digestion with Nt.BspQI, the nicked plasmids migrated at the open circular position. EcoRV digestion linearized the vector DNA, producing a single band on the gel. Addition of Vaccinia topoisomerase I resulted in gel-shift, suggesting that the enzyme was bound to the DNA. Some DNAs were hyper-linked (smeared appearance), or aggregated in the loading well. The enzyme-linked vector was recovered by PEG precipitation, removing most of the hyper-linked and aggregated DNAs. The recovery was 20–40% of the starting material. The present preparation is simpler than the method described by Heyman et al. [2], which required not only restriction digestions but also ligation of oligonucleotides, multiple phenol/chloroform extractions, and gel-purification of the vector DNA.


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)

Simplified preparation of Vaccinia topoisomerase I-linked vector.A. Schematic diagram of vector preparation. The procedure consisted of successive enzymatic reactions with a single precipitation step. The vector DNA was first digested with Nt.BspQI (5’-GCTCTTCN↓-3’, black arrows indicate the nicking sites) for 1 hr at 50°C, heated at 80°C for 20 min, and linearized with EcoRV (5’-GAT↓ATC-3’) for 1 hr at 37°C. These treatments produced two 24-nucleotide fragments (in green letters) which remained bound to the vector DNA (Tm 75.5°C). Purified Vaccinia topoisomerase I (5’-CCCTT↓-3’, red arrows indicate the nicking sites) was then added and linked with the vector DNA for 5 min at 37°C. Finally, the enzyme-linked vector was precipitated with 10% polyethyleneglycol (PEG, Mw 8,000) at 14,000 rpm for 20 min at 4°C. The whole process was completed in 3 hr. B. Agarose gel electrophoresis of the vector DNA at each step of preparation. The uncut vector was comprised of super coiled (SC) and open circular (OC) conformations (lane 2). Digestion with Nt.BspQI yielded nicked plasmids, which migrated at the open circular position (lane 3). EcoRV digestion linearized the vector DNA, producing a single band at 4.85 kb (lane 4). Addition of Vaccinia topoisomerase I resulted in band shift of the vector DNA (lane 5). Some DNAs were hyper-linked (smeared appearance) or aggregated in the loading well. Precipitation with 10% PEG recovered the enzyme-linked vector, but most of the hyper-linked and aggregated DNAs were removed (lane 6). Marker, λ/HindIII (the numbers indicate the size of DNA fragments in kbp).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139349.g004: Simplified preparation of Vaccinia topoisomerase I-linked vector.A. Schematic diagram of vector preparation. The procedure consisted of successive enzymatic reactions with a single precipitation step. The vector DNA was first digested with Nt.BspQI (5’-GCTCTTCN↓-3’, black arrows indicate the nicking sites) for 1 hr at 50°C, heated at 80°C for 20 min, and linearized with EcoRV (5’-GAT↓ATC-3’) for 1 hr at 37°C. These treatments produced two 24-nucleotide fragments (in green letters) which remained bound to the vector DNA (Tm 75.5°C). Purified Vaccinia topoisomerase I (5’-CCCTT↓-3’, red arrows indicate the nicking sites) was then added and linked with the vector DNA for 5 min at 37°C. Finally, the enzyme-linked vector was precipitated with 10% polyethyleneglycol (PEG, Mw 8,000) at 14,000 rpm for 20 min at 4°C. The whole process was completed in 3 hr. B. Agarose gel electrophoresis of the vector DNA at each step of preparation. The uncut vector was comprised of super coiled (SC) and open circular (OC) conformations (lane 2). Digestion with Nt.BspQI yielded nicked plasmids, which migrated at the open circular position (lane 3). EcoRV digestion linearized the vector DNA, producing a single band at 4.85 kb (lane 4). Addition of Vaccinia topoisomerase I resulted in band shift of the vector DNA (lane 5). Some DNAs were hyper-linked (smeared appearance) or aggregated in the loading well. Precipitation with 10% PEG recovered the enzyme-linked vector, but most of the hyper-linked and aggregated DNAs were removed (lane 6). Marker, λ/HindIII (the numbers indicate the size of DNA fragments in kbp).
Mentions: In the course of experiments, preparation of Vaccinia topoisomerase I-linked vector was streamlined. It consisted of successive enzymatic reactions with a single precipitation step, and the whole process was completed in 3 hr (Fig 4A). For preparation, the vector DNA was first digested with Nt.BspQI, linearized with EcoRV, and then linked with Vaccinia topoisomerase I followed by precipitation with 10% PEG. Fig 4B illustrates agarose gel electrophoresis of the vector DNA at each step of preparation. The uncut vector consisted of super-coiled and open circular forms. After digestion with Nt.BspQI, the nicked plasmids migrated at the open circular position. EcoRV digestion linearized the vector DNA, producing a single band on the gel. Addition of Vaccinia topoisomerase I resulted in gel-shift, suggesting that the enzyme was bound to the DNA. Some DNAs were hyper-linked (smeared appearance), or aggregated in the loading well. The enzyme-linked vector was recovered by PEG precipitation, removing most of the hyper-linked and aggregated DNAs. The recovery was 20–40% of the starting material. The present preparation is simpler than the method described by Heyman et al. [2], which required not only restriction digestions but also ligation of oligonucleotides, multiple phenol/chloroform extractions, and gel-purification of the vector DNA.

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