Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

“Combinatorial Strategy”: A Highly Efficient Method for Cloning Different Vectors with Various Clone Sites

Gang Zhang*, Anurag Tandon

Department of Medicine, Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada

Abstract: In this study, we generalized the “Combinatorial Strategy” for efficient cloning of different vectors with various clone sites. 1) Using originally existed clone sites from circular plasmids to prepare the inserts, if no appropriate sites available, performing SDM to create compatible sites, could achieve maximal correct digestion of the inserts. 2) Different vectors were digested with various restriction endonucleases, and then dephosphorylated after digestion. 3) Top10 competent cells were used for transformation to increase the transformant colonies. Our results showed that, when either blunt-sites or Xba I site was adopted for ligation, the percentages of positive clones were about 50%. Whereas, when different sites, including one blunt and another Pst I sites, Not I and Xho I sites, were used, the percentages of positive clones were nearly 100%. With this strategy, most vectors could be successfully cloned through “one ligation, one transformation, three to five minipreps”.

Keywords: DNA recombination, combinatorial strategy, vector cloning, site-directed mutagenesis, Top 10 cells, clone sites

Received March 22, 2013; Revised October 19, 2013; Accepted November 15, 2013

Citation: Gang Zhang, Anurag Tandon, “Combinatorial Strategy”: A Highly Efficient Method for Cloning Different Vectors with Various Clone Sites. American Journal of Biomedical Research, 1 (4). 112-119. January 2013. doi:10.12691/ajbr-1-4-7

1. Introduction

The successful production and intracellular replication of recombinant DNA were first reported in 1972 and 1973 [1,2], and these techniques have significantly revolutionized biomedical research in the past decades. At the genome era, more and more gene sequences of a growing number of species become available, and many different vector systems have been developed gradually. Therefore, there is an increasing need for efficient strategies to clone genes of interest into various expression vectors, precisely and efficiently, according to different aims. For example, to establish stable transgene cell lines, lentiviral vectors (LVs) might be good choices. And to achieve conditional transgene over-expression, drug-inducible systems were designed for this purpose [3,4,5,6,7,8,9,10,11,12].

Generally, the DNA recombination techniques were of low efficiency. To improve the efficiencies of plasmid vector cloning, recently, we reported an efficient method, “Combinatorial strategy”, for lentiviral vector cloning using site-directed mutagenesis (SDM) to insert clone site, BamH I [13]. SDM was first established at 1978 by Hutchison et al., and it is essential in gene functional studying, genetic engineering, protein engineering, and vector modifications [14,15,16]. Currently, the QuikChangeTM SDM System developed by Stratagene is a commonly adopted kit for mutagenesis using double-stranded plasmid DNAs as templates. The advantage of this strategy is that the products after mutagenesis are circular, double-stranded plasmid DNAs. As results, theoretically, 100% of the linearized DNA fragments are with correct-cleaved ends. Therefore, maximal efficiencies of ligation could be achieved [13].

In our neuroscience laboratory focused on Parkinson disease, we need to clone several different kinds of expression plasmid vectors for α-synuclein (α-Syn) and related genes, such as Rab, GDI, β5, Plk2 [17], etc., both wild type (WT) and mutants, to investigate their functional relations in vitro and in vivo. The aim of this study is to generalize our reported combinatorial strategy to efficiently construct different expression vectors with various clone sites, such as blunt-end sites (Swa I, Pme I and EcoR V), one blunt-end and one-overhang-end sites (Pme I or EcoR V, and Pst I), two different overhang-end sites (Not I and Xho I), and one overhang-end site (Xba I), and so on. Encouraged by our former report [13], with our efficient method, we successfully constructed 18 different vectors, mostly through “one ligation, one transformation, and a few minipreps”, for each of them. The percentages of positive clones with blunt sites, different clone sites, and a Xba I site, were approximately 47% ± 31% (n=13), 93% ± 12% (n=3), and 36% ± 10% (n=2), respectively. This study provided important quantitative models for efficient construction of different plasmid vectors, and laid significant foundations for gene functional analyses.

2. Materials and Methods

2.1. Design of SDM Primers

SDM was performed to insert Pst I sites for plasmids pcDNA4/β5WT and pcDNA4/β5T (Invitrogen, Lab stock) at the -end of β5WT and β5T open reading frames, respectively, to create pcDNA4/β5WT/-Pst I, and pcDNA4/β5T/-Pst I vectors. In addition, Xba I site was inserted by SDM at the and ends of β5T open reading frame, respectively, to create pcDNA4/β5T/-Xba I/-Xba I with pcDNA4/β5T as template. All primers, including Pst I and Xba I insertions, were designed according to the guide of Stratagene’s QuickChangeTM SDM kit, synthesized and purified by Integrated DNA Technologies. For all primers, mutagenized positions were denoted in lower case and underlined.

pcDNA4/-Pst I insertion forward: -CATCACCATTGAGTTTAAACctgcagCCGCTGATCAGCCTCGACTG;

pcDNA4/-Pst I insertion complement: -CAGTCGAGGCTGATCAGCGGctgcagGTTTAAACTCAATGGTGATG;

pcDNA4/-Xba I insertion forward: -GAATTCTGCAtctagaGATATCCAGC;

pcDNA4/-Xba I insertion complement: -GCTGGATATCtctagaTGCAGAATTC;

pcDNA4/-Xba I insertion forward: -CCATTGAGTTTAAACtctagaCCGCTGATCAGCCTC;

pcDNA4/-Xba I insertion complement: -GAGGCTGATCAGCGGtctagaGTTTAAACTCAATGG;

The melting temperatures (Tm, primer-to-template annealing temperature) were calculated by Online Software “Calculate a primer’s melting temperature for the QuickChange Site-Directed Mutagenesis Kit” created by David Kim (http://depts.washington.edu/bakerpg/primertemp.html), and primer-pair self-annealing temperatures (Tm*) were calculated by Integrated DNA Technologies SciTools OligoAnalyzer 3.1 (http://www.idtdna.com/analyzer/applications/oligoanalyzer/default.aspx), respectively (Table 1). All the DNA preparation kits, including Miniprep, Maxiprep, and Gel extraction kits, were purchased from QIAGEN. The DNA purities used in experiments were tested by NanoDrop-1000 Spectrophotometer (NanoDrop Technologies), and all the A260/280 values were ≥ 1.80.

Table 1. Characteristics of mutagenesis primer pairs

2.2. Mutagenesis

The PCR amplifications were carried out with GeneAmp® PCR System 2700 (AB Applied Biosystems). The 50µl PCR reactions were carried out with 50ng templates, 125ng of each forward and complement primers, 20µM of each dNTP, 2.5U of PfuUltra DNA polymerase in 1×reaction buffer (Stratagene). The thermal cycler program for amplifications was as follows: denaturation at for 2 min; 18 cycles at for 30 sec, annealing for 30 sec, at for pcDNA4/β5WT/-Pst I and pcDNA4/β5T/-Pst I insertions, for pcDNA4/β5WT/-Xba I and pcDNA4/β5T/-Xba I insertions, and for pcDNA4/β5T/-Xba I/-Xba I insertion, respectively (Table 2, Figure 2 and Figure 3), and at for 7 min; followed by a final extension at for 10 min. When the amplifications were finished, 1µl (10U) of Dpn I (Stratagene) was added into each reaction, and incubated at for 1 hour. Finally, 1µl reaction products were used for transformation into 50µl DH5α or Top10 competent cells (Invitrogen), respectively, according to the manufacturer’s guides. All the transformants were used to spread plates. Transformation colonies were selected and their plasmids were isolated by miniprep, and the positive mutants were identified by sequencing. All the primers are available upon request.

Table 2. Mutagenesis efficiency of Pst I and Xba I insertion

Figure 1. Schematic representation for the cloning of pWPI and pLenti LVs

a. α-Syn WT and A30P, A53T mutants were cleaved from pcDNAwith Pme I restriction endonuclease. Rab WT and T36N, Q mutants, and GDI WT and R218E, R mutants, were also digested with Pme I enzyme from pcDNA3.1. β5 WT and T mutant were cleaved from pcDNA4 with -EcoR V and -Pme I enzymes. The pWPI vector was digested with Swa I enzyme, followed by CIP treatment. EcoR V, Pme I and Swa I were blunt-end cleavage endonuclease enzymes, therefore, the inserts could be ligated into the pWPI vector.

b. Inducible vector pLenti (pLenti CMV/TO Puro DEST) was digested with EcoR V enzyme, and followed by CIP treatment, and then the inserts β5 WT and T were ligated with pLenti to clone the inducible LV pLenti/β5WT and pLenti/β5T.

Figure 2. Schematic representation for the cloning of pLVCT inducible LVs

The EGFP fragment could be cut off by -Pme I and -Pst I clone sites in the original pLVCT (pLVCT-tTR-KRAB) vector. To clone pLVCT/β5WT and pLVCT/β5T inducible vectors, a Pst I clone site was inserted at the -end of β5 WT and T mutant, respectively, taken pcDNA4/β5WT and pcDNA4/β5T plasmids as templates by SDM. Then the inserts were, at first, cleaved from pcDNA4 by EcoR V, and then by Pst I, sequentially. The pLVCT vector was treated with CIP following Pme I and Pst I digestion, and the EGFP fragment was replaced by β5 WT and T mutant, respectively.

Figure 3. Schematic representation for the cloning of inducible pTet/β5WT and pTet/β5T vectors

Two Xba I sites were sequentially inserted at the , and -ends of β5T, taken pcDNA4/β5T plasmid as template, by SDM to form pcDNA4/β5T/-Xba I/-Xba I. pcDNA4/β5WT plasmid was digested by Not I and Xho I, simultaneously, to recover 5'-Not I-β5WT-Xho I fragment. At the same time, pcDNA4/β5T/-Xba I/-Xba I plasmid was digested with Not I and Xho I, followed by the treatment with CIP, to recover dephosphorylated Not I-pcDNA4/-Xba I/-Xba I-Xho I vector fragment. And then, the insert and vector were ligated together to construct pcDNA4/β5WT/-Xba I/-Xba I vector. Finally, both of them were digested with Xba I to recover -Xba I-β5WT/T-Xba I inserts, and the pTet original vector was also cleaved by Xba I, followed by CIP treatment, to construct pTet/β5WTand pTet/β5T inducible expression vectors using Xba I clone site.

Figure 4. Comparison of the cloning efficiencies with various clone sites

The percentages of inserted colonies and positive colonies between Blunt sites/Different sites, Blunt sites/Xba I site, Different sites/Xba I site were analyzed by Mean ± SD and Student’s t-Test. The percentages of inserted clones with ligation of blunt-end sites, different clone sites, and a Xba I site, were approximately 77% ± 25% (n=13), 93% ± 12% (n=3), and 100% ± 0 (n=2), respectively. Among them, the efficiencies between blunt sites and Xba I site were significantly different (P=0.0064<0.05). Whereas, the differences between blunt sites/different sites and different sites/Xba I site were not significantly different (P=0.1304, and P=0.4226, P>0.05). On the other hand, the percentages of positive clones with ligation of blunt-end sites, different clone sites, and a Xba I site, were approximately 47% ± 31% (n=13), 93% ± 12% (n=3), and 36% ± 10% (n=2), respectively. Among them, the efficiencies between blunt sites/Xba I site and different sites/Xba I site were not significantly different (P=0.3468, and P=0.0154, P>0.05). In contrast, the efficiencies between blunt sites/different sites cloning were significantly different (P=0.0018<0.05).

2.3. Preparation of Vector and Insert DNA

LVs, pWPI/Neo (Addgene plasmid 12254, modified by replacing EGFP sequence with Neo) [13] was digested with Swa I (NEW ENGLAND BioLabs), pLenti CMV/TO Puro DEST (Addgene plasmid 17293) [18] was digested with EcoR V (NEW ENGLAND BioLabs), respectively. and pLVCT-tTR-KRAB (Addgene plasmid 11643) [9] was digested with Pme I and Pst I (NEW ENGLAND BioLabs), sequentially. In addition, pTetO-HGMoPrP (a kind gift from Dr. Prusiner, University of California, San Francisco) [12] was digested with Xba I (NEW ENGLAND BioLabs), and pcDNA4/β5T/-Xba I/-Xba I was digested with Not I and Xho I, simultaneously (NEW ENGLAND BioLabs) (Figure 1, Figure 2 and Figure 3; Table 3). All the digested vectors were treated with CIP (NEW ENGLAND BioLabs) at water bath for 1 hour, respectively, to remove the -phosphate groups [19]. And then, all the vectors were purified by 1% agarose gel electrophoresis to remove CIP, and recovered by QIAGEN Gel Extraction Kit [13].

Table 3. Construction efficiencies of different vectors with various clone sites

α-synuclein (Syn) WT and A30P, A53T mutants were cut off from pcDNA6 with Pme I. Rab WT, and T36N, Q mutants, GDI WT and R218E, R mutants were cut off from pcDNA3.1 with Pme I as well (Figure 1). The different DNA fragments of β5 WT and T mutant were cut off from pcDNA4 with EcoR V and Pme I, EcoR V and Pst I, Not I and Xho I, and Xba I, respectively, for different cloning (Figure 1, Figure 2 and Figure 3). Particularly, when β5WT and β5T mutant were severed from pcDNA4/-Pst I vectors with -EcoR V and -Pst I enzymes, at first, the vectors were digested by EcoR V enzyme, and then with Pst I enzyme, sequentially (Figure 2, Table 3). Because, there were a Pst I site at 952 nucleotide and a EcoR V site at 955 nucleotide. As a general rule, when the vectors and inserts were prepared with enzyme digestion, try to avoid using restriction enzymes that cleave within 12bp of each other in the multiple cloning site, or, after one of these sites has been cleaved, the second site will be located too close to the end of the linear DNA to allow efficient cleavage by the second enzyme [19]. All the vectors described above, except those particularly noted, were from lab stocks. Finally, all the inserts, α-Syn, Rab3A, GDI, and β5, both WT and mutants, were recovered by 1% agarose electrophoresis and gel extraction procedure, respectively.

2.4. Ligation

The ligation reactions were set up according to the molar ratios of inserts and vectors, and DNA amounts, indicated in Table 3. In 10µl ligation systems, the vector and insert were pooled together, warmed at for 5 min to melt any cohesive termini that have re-annealed during fragment preparation, then chilled on ice for 2 min, and then 1µl 10×T4 DNA ligase buffer and 0.5µl high-concentration T4 DNA ligase (400U/µl or 2000U/µl, NEW ENGLAND BioLabs) were added [19]. The 10µl reaction mixtures were incubated in GeneAmp® PCR System 2700 at for 16 hours followed by inactivation at for 10 min, and then set at until transformation.

2.5. Transformation and Identification

2µl (about 30~80ng) volumes of the ligation products were used to transform 50µl Top10 competent cells according to the manufacturer’s instructions (Table 3). In order to obtain more colonies, all the transformation cells were used to spread plates. Positive colonies were primarily analyzed by restriction endonuclease digestion, and further confirmed by DNA sequencing.

2.6. Data statistics

Data were analyzed by Mean ± SD and Student’s t-Test.

3. Results

3.1. Pst I and Xba I insertion by SDM

SDM was performed by PCR, and the parent template DNAs were digested with Dpn I. Different annealing temperatures were used for pcDNA4/β5WT/-Pst I, pcDNA4/β5T/-Pst I, pcDNA4/β5TW/-Xba I, pcDNA4/β5T/-Xba I, and pcDNA4/β5T/-Xba I/-Xba I insertions, representing approximately Tm*, Tm + , and Tm* (Table 1 and Table 2; Figure 2 and Figure 3), respectively. After transformation into Top10 or DH5α cells and identified by sequencing, our results showed that, the mutagenesis efficiencies were approximately 50% for pcDNA4/β5WT/-Pst I insertion, 33.3% for pcDNA4/β5T/-Pst I insertion, 10% for pcDNA4/β5T/-Xba I insertion, and 50% for pcDNA4/β5T/-Xba I/-Xba I insertion, respectively. In addition, positive mutagenesis clones were not obtained for pcDNA4/β5WT/-Xba I insertion after sequencing 18 minipreps (Table 1 and Table 2). These data demonstrated that the majority of our designed mutagenesis was successfully achieved.

3.2. Efficient Cloning of Different Vectors with Various Clone Sites

3.2.1. Construction of LVs with Blunt-end Clone Sites

A blunt-end clone site Swa I of pWPI/Neo LV was employed as clone site. To protect the vector self-circularization, we removed the -phosphate groups of the vector DNA with calf intestinal phosphatase (CIP) treatment following Swa I digestion. This treatment could diminish the background of transformed colonies that carried empty plasmids. At the same time, the colony number would be significantly decreased after transformation [19,20,21]. Insert (α-Syn WT and A30P, A53T mutants; Rab WT and T36N, Q mutants; GDI WT and R218E, R mutants; β5 WT and T mutant; respectively) and pWPI/Neo vector DNAs were pooled together in 10µl ligation reactions with DNA concentrations around 18.9~42ng/µl, and insert to vector molar ratios about 1~3.5:1 (Table 3, Figure 1). To improve the transformation efficiencies, Top10 cells were used as hosts, and 2 to about 20 colonies were obtained after transformation. After identification by restriction endonulease digestion and sequencing, the percentages of positive clones with monomeric, correct-oriented inserts were ranging from 20% to 100%, respectively (Table 3). Except the cloning of pWPI/GDIWT/Neo, all the blunt-end cloning described above was achieved by performing one ligation and one transformation. For the cloning of pWPI/GDIWT/Neo, 15 colonies were resulted from the first ligation and transformation, and after identification with BamH I digestion, 9 clones were with opposite-directed inserts, and the other 6 clones were with unknown DNAs. Through the second ligation and transformation, positive clones of pWPI/GDIWT/Neo were obtained (Table 3).

To construct inducible LVs, pLenti CMV/TO Puro DEST was digested with EcoR V, and followed by CIP treatment. In 10µl ligation system, the molar ratios of insert β5 WT and T mutant to pLenti vector were 22:1 and 14.1:1, and the DNA amounts were about 18.9ng/µl and 14ng/µl, respectively (Table 3, Figure 1). After transformation into Top10 cells, 12 and 13 colonies were obtained, and the percentages of positive clones were 37.5% and 12.5%, respectively. Taken together, the percentages of inserted clones and positive clones with blunt-end ligations, were approximately 77% ± 25%, and 47% ± 31%, respectively (Table 3, Figure 4).

3.2.2. Construction of Different Vectors with Various Clone Sites

pLVCT-tTR-KRAB was first digested with Pme I, then with Pst I, sequentially, and then treated with CIP. In 10µl ligation system, the molar ratios of inserts β5WT and T mutant to pLVCT vector were around 3:1, and the DNA amounts were around 17.6ng/µl, respectively (Table 3, Figure 2). After transformation into Top10 cells, about 300 and 100 colonies were obtained. After identification, all the recombinant clones were with correct directions, therefore, the percentages of inserted clones and positive clones were both 80% and 100%, respectively (Table 3).

Due to the failure to insert -Xba I site for pcDNA4/β5WT (Table 2), pcDNA4/β5T/-Xba I/-Xba I was digested with Not I and Xho I, simultaneously, and followed by CIP treatment, the insert β5WT (digested with Not I and Xho I) was ligated into the vector pcDNA4/-Xba I/-Xba I to form pcDNA4/β5WT/-Xba I/-Xba I (Figure 3). The molar ratio of insert β5WT to pcDNA4/-Xba I/-Xba I vector was about 1.9:1, and the DNA amount was around 28.1ng/µl, respectively. After transformation into Top10 cells, about 500 colonies were obtained, the percentages of inserted clones and positive clones were both 100% (Table 3).

3.2.3. Construction of Inducible pTet/β5WT and pTet/β5T Vectors with a Unique Xba I Clone Site

Previously, we reported the construction of pWPI/hPlk2 WT and mutant vectors with a BamH I site by inserting BamH I clone site at the -ends of hPlk2 [13]. The pTetO-HGMoPrP vector was digested with Xba I to cut off the insert HGMoPrP, followed by CIP treatment. β5WT and β5T inserts were cleaved from vectors pcDNA4/-Xba I/-Xba I, and ligated with pTet, to form inducible vector pTet/β5WT and pTet/β5T (Figure 3). In 10µl ligation system, the molar ratios of insert β5WT and T mutant to pTet vector were about 2.1:1 and 1.5:1, and the DNA amounts were about 21.9ng/µl and 19.7ng/µl, respectively. After transformation into Top10 cells, about 1000 colonies of each were obtained. The percentages of inserted clones were 100% for both of them, and those of positive clones were 28.6% and 42.9%, respectively (Table 3).

Taken together, among the identified clones, the percentages of inserted clones and positive clones with ligation of blunt-end sites (including Swa I, Pme I and EcoR V), different clone sites (EcoR V or Pme I and Pst I; Not I and Xho I), and a Xba I site (Figure 1, Figure 2 and Figure 3), were approximately 77% ± 25% and 47% ± 31% (n=13), 93% ± 12% and 93% ± 12% (n=3), 100% ± 0 and 36% ± 10% (n=2), respectively. Among them, the efficiencies of blunt-end cloning and Xba I site cloning were not significantly different (47% ± 31% vs 36% ± 10%, P>0.05), these data were similar with our previous report with a BamH I clone site¹³ (Table 3). In contrast, the efficiencies between blunt-end cloning and different site cloning (47% ± 31% vs 93% ± 12%), different site cloning and Xba I site cloning (93% ± 12% vs 36% ± 10%) were significantly different (P<0.05).

4. Discussion

In our former report, SDM was performed to insert BamH I clone site for the inserts, hPlk2 WT and mutants and EGFP gene, to efficiently construct LVs [13]. In this study, Pst I clone site at the -ends of β5 WT and T mutant genes was inserted by SDM, to achieve the construction of inducible LVs, pLVCT/ β5WT and pLVCT/ β5T1A, with -blunt end and -Pst I as clone sites. In addition, Xba I site was also inserted at the - and - ends of β5T mutant, respectively, to construct the inducible expression vector pTet/β5WT and pTet/β5T1A (Table 1 and Table 2). The Stratagene SDM protocol suggested that the prime pairs should be of 25~45 bases in length with melting temperatures (Tm) ≥78˚C. Compared with our previous report [13], in this study, prime pairs with shorter length (ranging from 26 to 46 bases) and lower Tm (ranging from 49.8 to 75.2˚C), were adopted for SDM (Table 1). And the efficiencies of mutagenesis were lower (from 10% to 50%) than our former report (Table 2). These data suggested that longer prime pairs with higher Tm might be more efficient for clone site insertion than shorter ones [13] (Table 1 and Table 2). Although SDM is a powerful tool to change DNA sequences at specific positions in genetic engineering and clone site insertion, under some circumstances, the QuickChangeTM SDM system of Stratagene could lead to lower PCR efficiency, and even the failure of mutagenesis. In these cases, some modifications would be made to circumvent the problems, such as using primers containing extended non-overlapping sequences at the 3’-end and primer-primer complementary sequences at the 5’-end, running two PCR reactions in parallel with each one of the forward and reverse primers, and so on [13,15,16,23]. After the insertion of Pst I clone site at the 3’-ends of β5WT and β5T1A mutant with SDM, 100% of the linearized 5’-EcoR V-β5WT/T1A-3’-Pst I DNA fragments were theoretically with correct cutting ends. Whereas, due to the insufficient stable-association of the restriction endonucleases with the extra 3-4 bases at the terminal restriction sites, correct cutting ends could not be confirmed with the method of incorporating of restriction sites into PCR primers [24,25]. Our data demonstrated that the clone efficiencies were significantly improved with this strategy. The percentages of positive clones with monomeric, correct-oriented inserts were 80% for pLVCT/β5WT, and 100% for pLVCT/β5T, respectively (Table 3, Figure 2). After Xba I site was inserted at the - and - ends of β5T mutant, respectively, the Tet-inducible expression vectors, pTet/β5WT and pTet/β5T, were successfully constructed with a unique Xba I clone site. The percentages of positive clones were 28.6% and 42.9% (36% ± 10%, n=2), respectively (Table 3, Figure 3). These data were in agreement with our former report [13].

Ligation is the critical step in plasmid vector cloning, and the number of transformants is radically affected by the ligation efficiencies. Removing the -phosphate residues from both termini of the vector DNA can efficiently minimize the re-circularization of vector DNA, and therefore, decrease the background with empty transformants [19]. At the same time, the transformation colonies were significantly decreased, particularly for ligation with blunt ends, because the ligation efficiency of blunt ends was much lower than cohesive end ligation [26,27]. Our data demonstrated this phenomenon. In our experiment, the transformant number of blunt-end ligations was from to 20, whereas, the transformation colonies of the ligation with one blunt end and one cohesive end were about 100 and 300, respectively. Furthermore, about 500 colonies were obtained when Not I and Xho I sites were used for ligation, and about 1000 colonies were resulted from Xba I site ligation (Table 3). Finally, the ligation efficiencies were significantly affected by vector sizes. For instance, when pLVCT (12kb), pcDNA(5.1kb), and pTet (3.2kb) were used for ligation with β5 WT and T mutant inserts, more transformation colonies were obtained with the decrease of vector sizes (Table 3). To investigate the effects of the molar ratios of insert to vector on the transformant number and the percentages of positive clones, a large range of molar ratios, from 1.7 to 22:1, was used for ligation with blunt ends, there was no significant different between the number of obtained transformants and the percentages of positive clones (Table 3). Taken together, the percentages of positive clones with the ligation of blunt ends, different clone sites, and a Xba I, were approximately 47% ± 31% (n=13), 93% ± 12% (n=3), and 36% ± 10% (n=2) of identified clones, respectively. The efficiencies of blunt-end cloning and Xba I site cloning were not significantly different (47% ± 31% vs 36% ± 10%, P>0.05), In contrast, the efficiencies between blunt-end cloning and different site cloning (47% ± 31% vs 93% ± 12%), different site cloning and Xba I site cloning (93% ± 12% vs 36% ± 10%) were significantly different (P<0.05). The data of the cloning with a Xba I site were consistent with those of the cloning with a BamH I site (Table 3) [13].

In our previous study, we reported a combinatorial method for efficient cloning of LVs, specifically taking pWPI/Neo as vector backbone, hPlk2 WT, mutants, and EGFP gene as inserts, and BamH I as clone site [13]. In order to generalize our efficient strategy, in this study, we reported quantitative models for efficient construction of different plasmid vectors, which adopted different vectors and inserts, and various clone sites. The sizes of the vector backbones were from about 3.2 to 12kb, and those of the inserts were from about 0.64 to 1.44kb (Table 3). Taken together with our former reported hPlk2 gene (about 2.1kb) [13], the size range of the inserts included most of the protein coding open reading frames. Our data demonstrated that the combinatorial method was applicable for almost all the plasmid vector cloning with various clone sites. We designated our efficient method as “Combinatorial Strategy”. With this Strategy, most vectors could be successfully constructed through “one ligation, one transformation, and three to five minipreps”. This study provided important quantitative models for efficient cloning of different vectors with various clone sites, and could radically facilitate the functional analyses of genes of interest in vivo and in vitro.

Competing Interests

The authors declare they have no competing interests.

Acknowledgement

We thank Dr. Didier Trono and Dr. Patrick Aebischer for the original pWPI and pLVCT-tTR-KRAB vectors, Dr. Stanley B. Prusiner for the original pTetO-HGMoPrP vector, Dr. Eric Campeau for the original pLenti CMV/TO Puro DEST vector, Robert Strome for the useful discussions, Yan Liang for the sequencing. This work was supported by Canadian Institutes of Health Research (CIHR) grant MOP84501 to AT and the Parkinson Society of Canada.

Author Contributions

G.Z. and A.T. conceived and designed the experiments. G.Z. performed the experiments and carried out data analysis. G.Z. wrote the paper.

References

[1]   | Jackson, D.A., Robert, H.S. and Berg, P., Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of E. coli, Proc. Nat. Acad. Sci. USA, 69: 2904-2909, 1972.
[2]   | Cohen, S.N., Chang, A.C.Y., Boyert, H.W. and Hellingt, R.B., Construction of biologically functional bacterial plasmids in vitro, Proc. Nat. Acad. Sci. USA, 70: 3240-3244, 1973.
[3]   | Naldini, L., Blömer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., Verma, I.M. and Trono, D., In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science, 272: 263-267, 1996.
[4]   | Cui, Y., Golob, J., Kelleher, E., Ye, Z., Pardoll, D., Cheng, L., Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells, Blood, 99: 399-408, 2002.
[5]   | Lois, C., Hong, E.J., Pease, S., Brown, E.J., Baltimore, D., Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors, Science, 295: 868-872, 2002.
[6]   | Pfeifer, A., Ikawa, M., Dayn, Y., Verma, I.M., Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos, Proc Natl Acad Sci USA, 99: 2140-2145, 2002.

[7]  
Yu, X., Zhan, X., D’Costa, J., Tanavde, V.M., Ye, Z., Peng, T., Malehorn, M.T., Yang, X., Civin, C.I. and Cheng, L., Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells, Mol Ther, 7: 827-838, 2003.

[8]   | Zufferey, R., Dull, T., Mandel, R.J., Bukovsky, A., Quiroz, D., Naldini, L., and Trono, D., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery, J Virol, 72: 9873-9880, 1998.
[9]   | Szulc, J., Wiznerowicz, M., Sauvain, M.O., Trono, D., Aebischer, P., A versatile tool for conditional gene expression and knockdown, Nat Methods, 3: 109-116, 2006.
[10]   | Hotta, A., Cheung, A.Y., Farra, N., Vijayaragavan, K., Séguin, C.A., Draper, J.S., Pasceri, P., Maksakova, I.A., Mager, D.L., Rossant, J., Bhatia, M. and Ellis, J., Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency, Nat Methods, 6: 370-376, 2009.
[11]   | Sommer, C.A., Stadtfeld, M., Murphy, G.J., Hochedlinger, K., Kotton, D.N. and Mostoslavsky, G., Induced pluripotent stem cell generation using a single lentiviral stem cell cassette, Stem Cells, 27: 543-549, 2009.

[12]  
Tremblay, P., Meiner, Z., Galou, M., Heinrich, C., Petromilli, C., Lisse, T., Cayetano, J., Torchia, M., Mobley, W., Bujard, H., DeArmond, S.J. and Prusiner, S.B., Doxycycline control of prion protein transgene expression modulates prion disease in mice, Proc Natl Acad Sci USA, 95: 12580-12585, 1998.

[13]   | Zhang, G. and Tandon, A., Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site, Sci Rep, 2: 415, 2012.
[14]   | Hutchison, C.A.III., Phillips, S., Edgell, M.H., Gillam, S., Jahnke, P. and Smith, M., Mutagenesis at a specific position in a DNA sequence, J Biol Chem, 253: 6551-6560, 1978.
[15]   | Norrander, J., Kempe, T. and Messing, J., Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis, Gene, 26: 101-106, 1983.
[16]   | Liu, H. and Naismith, J.H., An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol, BMC Biotech, 8: 91, 2008.
[17]   | Visanji, N.P., Wislet-Gendebien, S., Oschipok, L.W., Zhang, G., Aubert, I., Fraser, P.E. and Tandon, A., The effect of S129 phosphorylation on the interaction of α-synuclein with synaptic and cellular membranes, J Biol Chem, 286: 35863-35873, 2011.
[18]   | Campeau, E., Ruhl, V.E., Rodier, F., Smith, C.L., Rahmberg, B.L., Fuss, J.O., Campisi, J., Yaswen, P., Cooper, P.K. and Kaufman, P.D., A versatile viral system for expression and depletion of proteins in mammalian cells, PLoS One, 4: e6529, 2009.
[19]   | Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Plainview, NY, 2001.
[20]   | Seeburg, P., Shine, J., Martial, J.A., Baxter, J.D., Goodman, H.M., Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone, Nature, 270: 486-494, 1977.
[21]   | Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J. and Goodman, H.M., Rat insulin genes: construction of plasmids containing the coding sequences, Science, 196: 1313-1319, 1977.
[22]   | Marumoto, T., Tashiro, A., Friedmann-Morvinski, D., Scadeng, M., Soda, Y., Gage, F.H. and Verma, I.M., Development of a novel mouse glioma model using lentiviral vectors, Nat Med, 15: 110-116, 2009.
[23]   | Edelheit, O., Hanukoglu, A. and Hanukoglu, I., Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies, BMC Biotech, 9: 61, 2009.
[24]   | Jung, V., Pestka, S.B. and Pestka, S., Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites, Nucleic Acids Res, 18: 6156, 1990.
[25]   | Testori, A., Listowsky, I. and Sollitti, P., Direct cloning of unmodified PCR products by exploiting an engineered restriction site, Gene, 143: 151-152, 1994.
[26]   | Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Plainview, NY, 1989.
[27]   | Yang, S., Li, X., Ding, D., Hou, J., Jin, Z., Yu, X., Bo, T., Li, W. and Li, M., A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase, Biotechnol Appl Biochem, 42: 223-226, 2005.