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Protein Expr. Purif. 47, 562–570. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. 2006

Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.

Notes: In this study, the Flexi® Vector Systems was compared with the Gateway® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway® vector and the equivalent Flexi® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi® Vector system was shorter at 6–8 days compared to 12 days for the Gateway® system. They also found the Flexi® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil® PCR Clean-Up System or Wizard® SV 96 PCR Clean-Up, ligated into the Flexi® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi® Vectors and transfer of cDNA inserts between different Gateway® vectors and found similar performance in the two systems. For the Flexi® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi® Vectors. (3533)

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Hum. Mol. Genet. 15, 999–1013. An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. 2006

Disset, A., Bourgeois, C.F., Benmalek, N., Claustres, M., Stevenin, J. and Tuffery-Giraud, S.

Notes: To construct dystrophin minigenes, genomic DNA containing a mutation in dystrophin was amplified for exons 30, 31 and 32. The three PCR fragments were combined and amplified into one product. This overlap-extension PCR generated two minigenes which were then cloned into the pGEM®-T Vector and sequenced. After EcoR I digestion, the minigenes were ligated into the pSI Mammalian Expression Vector and transiently transfected into C2C12 cells for expression analysis. (3499)

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Nucl. Acids Res. 34, 584-592. Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence. 2006

Nehdi, A. and Perreault, J.P.

Notes: To characterize sequence variability of the catalytic center of the hepatitis delta virus (HDV) ribozyme, which includes a self-cleaving motif, the authors developed an in vitro selection process that allowed them to select self-cleaving sequence variants from a pool of HDV ribozymes. The selection process started with a library of DNA oligonucleotides corresponding to the HDV ribozyme sequence that had been randomized at a 25-nucleotide sequence. The oligos contained known sequences at the 5´ and 3´ ends to allow amplification. The library was amplified by PCR, then transcribed. The resulting RNA was separated by polyacylamide gel electrophoresis, and the self-cleaved transcripts were excised, purified and reverse transcribed. A 3´ extension using terminal deoxynucleotidyl transferase added a known sequence to the 3´ end so that the resulting cDNA could by amplified by PCR. Thus another cycle of selection could begin. The PCR products generated at the start of the process and at each cycle were cloned into the pGEM®-T Vector. (3467)

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Nucl. Acids Res. 34, e67. GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. 2006

Buzdin, A., Kovalskaya-Alexandrova, E., Gogvadze, E. and Sverdlov, E.

Notes: The authors selected repetitive elements in the human genome using a novel technique: GREM. T4 DNA Ligase was used to ligate adapters to digested genomic DNA prior to PCR, and exonuclease III was used to generate the necessary 5´ termini. After the final amplification, the PCR products were cloned into the pGEM®-T Vector, then sequenced. (3550)

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Am. J. Pathol. 168, 706–713. Expression of Pax2 in human renal tumor-derived endothelial cells sustains apoptosis resistance and angiogenesis. 2006

Fonsato, V., Buttiglieri, S., Deregibus, M.C., Puntorieri, V., Bussolati, B. and Camussi, G.

Notes: To further understand the transcription factor paired-box 2 (PAX2) gene, 2µg of total RNA isolated from Kaposi’s sarcoma cells were reverse transcribed, and 5µl of the RT reaction was amplified. The PCR product was then cloned into the pTARGET™ Mammalian Expression Vector and two clones identified: PAX2 in the sense orientation and a second in the antisense orientation. The clones were then transfected into two different cell lines: the antisense PAX2 into renal tumor-derived endothelial cells where PAX2 is expressed, and the sense into normal human microvascular endothelial cells where PAX2 is not normally expressed. The effects of PAX2 expression on the cell lines were then examined. (3496)

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Mol. Cell. Biol. 17, 1331-1343. The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination. 2006

Wijeratne, A.J., Chen, C., Zhang, W., Timofejeva, L. and Ma, H.

Notes: The authors identify PARTING DANCERS as a gene involved in male meiosis in Arabidopsis using a microarray generated from meiotic-stage anthers. To confirm the sequence obtained by microarray analysis, RT-PCR was performed and the resulting cDNA was cloned into the pGEM®-T Vector and sequenced. To generate probes for in situ hybridization, fragments of ptd were amplified, cloned into the pGEM®-T Vector, then labeled with digoxygenin through in vitro transcription. (3468)

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Clin. Can. Res. 12, 2032-37. Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA. 2006

Wu, S., Wang, S., Wang, W., Xi, L., Tian, X., Chen, G., Wu, Y., Zhou, J., Xu, G., Lu, Y. and Ma, D.

Notes: The coding sequence of the Human Papilloma Virus (HPV16) E7 oncogene was isolated following total RNA purification from CaSki cells, RT-PCR and PCR and then cloning into the pGEM®-T Easy vector. In order to test the effectiveness of antisense HPV16 E7 therapy against cervical cancer, an adeno-associated virus vector was used to transfer the antisense construct of the E7 coding sequence into CaSki cervical cancer cells. (3395)

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Appl. Environ. Microbiol. 72, 2691–2706. Oligonucleotide array for identification and detection of pythium species. 2006

Tambong, J.T., de Cock, A.W., Tinker, N.A. and Levesque, C.A.

Notes: This study compared detection of Pythium species in soil samples by DNA array hybridization and PCR cloning. Three Pythium species were amplified from soil samples, a single 3´ A was added to the resulting PCR product, and the DNA was ligated into the pGEM®-T Easy Vector at 4°C overnight. After the ligation was transformed into JM109 Competent Cells, and 100 colonies were chosen and grown overnight in LB broth. The plasmid DNA was isolated using the Wizard® SV 96 Plasmid DNA Purification System and then sequenced. (3437)

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Appl. Environ. Microbiol. 72, 3274–3283. Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity. 2006

Cimerman, A., Arnaud, G. and Foissac, X.

Notes: To purify stolbur phytoplasma DNA from total DNA of infected periwinkle plants, two rounds of suppression subtractive hybridization (SSH) were performed, followed by amplification with Taq DNA polymerase. The resultant PCR products (1µl) were ligated into 50ng of pGEM®-T Easy Vector using 3 units T4 DNA Ligase. After transformation of DH10B cells, ampicillin-resistant colonies were grown and the plasmids purified using the Wizard® Plus SV Minipreps DNA Purification System. The insert lengths were estimated after EcoR I digestion and agarose gel electrophoresis prior to amplification and labeling with digoxigenin. These probes were used for dot hybridization with denatured healthy or infected plant DNA (10µg) and the corresponding plasmid as a positive control (100 ng). (3436)

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J. Biol. Chem. 281, 13915–13921. NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling. 2006

Suzuki, A., Ogura, T. and Esumi, H.

Notes: A deletion mutation of the serine/threonine protein kinase NDR2 was created by PCR using two mutagenesis primers and two plasmid-based primers. After amplification, the two products were run on a 1% agarose gel and extracted using the Wizard® SV Gel and PCR Clean-Up System. The purified fragments were mixed, annealed, re-amplified and then digested prior to cloning into an expression vector. The human colorectal cancer cell lines HCT-116, DLD-1, and SW480 used in the study were seeded into a 24-well plate at 5 × 104/well, and transfected using the TransFast™ Transfection Reagent. The transfection was assessed with a green fluorescent protein expression vector. (3438)

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J. Biol. Chem. 281, 17410-17419. ATP binding to a unique site in the type-1 S2- inositol 1,4,5-triphosphate receptor defines susceptibility to phosphorylation by protein kinase A. 2006

Wagner, L.E., Betzenhauser, M.J. and Yule, D.I.

Notes: N-terminal GST fusion proteins of portions of the type-1 S2- Ionsitol 1,4,5-triphosphate receptor were created using the pFN2A (GST) Flexi® Vector. The constructs were expresed in BL21 (DE3) pLysS cells and used for ATP-binding assays. (3392)

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FEBS Lett. 580, 755-762. Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). 2006

Lee, S.J., Jang, B.C., Lee, S.W., Yang, Y.I., Suh, S.I., Park, Y.M., Oh, S., Shin, J.G., Yao, S., Chen, L. and Choi, I.H.

Notes: Many cancer cells upregulate the co-signaling molecule B7-H1, confering resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System. (3451)

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Plant Physiol. 137(2), 557–66. Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii. 2005

Rymarquis, L.A., Handley, J.M., Thomas, M. and Stern, D.B.

Notes: These authors describe map-based cloning tools for Chlamydomonas reinhardtii, (green yeast). In this case-based study of existing and projected resources for C. reinhardtii mapping, mcd4 and mcd5 mutants were mapped by crossing to the infertile strain known as Chlamydomonas grossii, S1-D2, with several known marker sites. To reduce the time and expense of mapping, bulked segregant analysis (BSA) and marker duplexing were evaluated. In BSA, DNAs from multiple segregating progeny (up to six in this study) are combined, and results from PCR-based markers are examined for significant bias from a roughly equal contribution from each parent. This case study used 57–72 markers to span the 1,107-cM genome using approximately 3,350 PCR amplifications. The researchers picked single colonies from a plate and placed each colony in a hypotonic EDTA solution. After boiling for 5 minutes, the supernatant was collected by centrifugation and the DNA quantitated. Twenty nanograms of DNA thus isolated was used as the template for PCR. The reaction included GoTaq® DNA polymerase with the enhancers 8.5%glycerol and 0.83% formamide in a 30µl reaction. Forty cycles of amplification were performed and the results were analyzed by agarose gel electrophoresis. The various amplification reactions included the use of BSA and both monoplex and duplex reactions with amplicons of 90bp–625bp. (3279)

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J. Bacteriol. 187, 3762-3778. Sulfur amino acid metabolism and its control in Lactococcus lactis IL1403. 2005

Sperandio, B., Polard, P., Ehrlich, D.S., Renault, P. and Guédon, E.

Notes: To study the transcription levels of several genes involved in cysteine and methionine metabolism, Lactococcus lactis strains deficient in these genes were created by double-crossover homologous integration. To do so, PCR was performed to amplify sequences upstream and downstream of the target gene, and the PCR products were ligated and cloned into the pGEM®-T Easy Vector. The resulting vectors were fused to pGhost9, a vector designed for homologous recombination in L. lactis. Once the gene deletions were verified, quantitative real-time RT-PCR was performed to quantitate expression levels of a variety of genes involved in Cys and Met metabolism. (3461)

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Nucl. Acids Res. 33, 4762-4774. Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe. 2005

Gonzalez-Barrera, S., Sanchez, A., Ruiz, J.F., Juarez, R., Picher, A.J., Terrados, G., Andrade, P. and Blanco, L.

Notes: An open reading frame encoding a putative DNA polymerase, SpPol4, was amplified from S. pombe genomic DNA by PCR. The resulting PCR product was cloned into the pGEM®-T Easy Vector, and the sequence was verified. Based on sequence analysis, the authors hypothesize that SpPol4 has deoxyribose phosphate (dRP) lyase activity, suggesting that the enzyme plays a role in base excision repair. The authors perform a dRP lyase activity assay with an oligonucleotide substrate labeled using [32P]ddATP and terminal deoxynucleotidyl transferase. (3465)

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Infect. Immun. 73, 2611-2620. Molecular cloning and characterization of three beta-defensins from canine testes. 2005

Sang, Y., Ortega, M.T., Blecha, F., Prakash, O. and Melgarejo, T.

Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM®-T Vector, which was then linearized and treated with exonuclease III to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)

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DNA Research 12, 257–267. Preparation of a Set of Expression-Ready Clones of Mammalian Long cDNAs Encoding Large Proteins by the ORF Trap Cloning Method. 2005

Nakajima, D., Saito, K., Yamakawa, H., Kikuno, R.F., Nakayama, M., Ohara, R., Okazaki, N., Koga, H., Nagase, T. and Ohara, O.

Notes: To prepare expression-ready cDNA clones of 1589 putative full-length ORFs (from human and mouse genes) with an average size of 2.8 kb, a linear trap vector was created. Generated by PCR, this linear plasmid contained gene-specific sequences to allow homologous recombination. In addition, 5–10µg of a plasmid containing the same gene-specific sequence and the linear trap vector were purified using the Wizard® SV 96 PCR Clean-Up System. The purified DNA was resuspended in water and transformed into E. coli cells. The plasmid purified after the recombination was transferred to a Gateway expression vector and 100–200ng expressed in the TNT® T7 Quick Coupled Reticulocyte Lysate System with 0.2µl of FluoroTect™ GreenLys tRNA. The proteins expressed were resolved using SDS-PAGE. (3439)

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Mol. Cell. Biol. 25, 2861-2870. Assembly of the mariner Mos1 synaptic complex. 2005

Auge-Gouillou, C., Brillet, B., Hamelin, M.H., and Bigot, Y.

Notes: A transposase protein (Tnp) open reading frame (amino acids 1 to 345) was amplified from the Drosophila mauritiana Mos1 mariner transposable element using GoTaq® DNA polymerase. The Tnp open reading frame was then cloned with into the pGEM®-T Easy Vector and sequenced before further subloning. Tnp protein was purified and used in gel-shift assays to demonstrate Tnp binding regions in the Mos1 transposable element. (3344)

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J. Biol. Chem. 280, 28215-28220. Determination of the functionality of common APOA5 polymorphisms. 2005

Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

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Plant Physiol. 135, 1540–1551. Isolation and characterization of a TERMINAL FLOWER homolog and its correlation with juvenility in citrus. 2004

Pillitteri, L.J., Lovatt, C.J. and Walling, L.L.

Notes: The authors identified a TERMINAL FLOWER homolog, CsTFL, in Washington navel oranges (Citrus sinensis) and investigated its role and the role of other genes in juvenility and flower production. The CsTFL gene was amplified from genomic DNA using PCR and degenerate primers, and amplification products were cloned into the pGEM®-T Easy Vector. The resulting clones were sequenced using the fmol® DNA Cycle Sequencing System. The CsTFL cDNA was amplified by RT-PCR, using 4 µg of total RNA from whole flowers and ImProm-II™ Reverse Transcriptase. The amplified cDNA was then cloned into the pGEM®-T Easy Vector. To evaluate CsTFL gene copy number and allele origins, the authors performed a Southern blot with 10 µg of genomic DNA and a CsTFL probe labeled with the Prime-a-Gene® Labeling System. To characterize CsTFL expression in various citrus tissues, RT-PCR was performed with 2 µg of total RNA and ImProm-II™ Reverse Transcriptase. The levels of CsTFL RNA and other RNAs were determined by cDNA synthesis using ImProm-II™ Reverse Transcriptase, followed by real-time PCR. Amplification products were quantitated by SYBR® Green fluorescence. Standard curves for each real-time PCR target were generated using known amounts of in vitro transcribed RNA. Prior to reverse transcription and real-time PCR, total RNA samples were treated with RQ1 RNase-Free DNase to remove DNA contaminants. (3650)

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Biochem. Biophys. Res. Commun. 321(1), 259-265. Cloning of hOST-PTP: the only example of a protein-tyrosine-phosphatase the function of which has been lost between rodent and human 2004

Cousin, W., Courseaux, A., Ladoux, A., Dani, C., and Peraldi, P.

Notes: Researchers used GoTaq® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard® SV Gel and PCR Clean-Up System prior to cloning into the pGEM®-T Easy Vector.  The Prime-a-Gene® Labeling System was used to make 32P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

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RNA 10, 277–286. Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. 2004

Verge´, V., VonLanthenm, M., Masson, J.M., Trachsel, H., and Altmann, M.

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM®-T Vector. (2845)

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J. Gen. Virol. 85(Pt 7), 2111-21. Molecular characterization of Penicillium chrysogenum virus reconsideration of the taxonomy of the genus Chrysovirus. 2004

Jiang, D. and Ghabrial, S.A.

Notes: The authors cloned and sequenced the dsRNA from Penicillium chrysogenum virus (PcV) and analyzed the gene products of the segmented virus. After reverse transcription followed by PCR to confirm the 5’ and 3’ ends of the dsRNA segments, RT-PCR was used to amplify the complete cDNAs for the four viral segments. The products were cloned into pGEM®-T Vector after A-tailing. To confirm the ORFs predicted for each of the four segments, the cDNAs were added to TNT® T7 Quick Coupled Transcription/Translation System reactions that included radiolabeled methionine. The resulting proteins ranged in size from 94-128 kDa and were separated on an 8% SDS-PAGE gel and analyzed by autoradiography. Gradient-purified PcV virions were digested with Sequencing Grade Modified Trypsin at 37°C for 18 hours and the digestion products were separated by reverse-phase HPLC on a C18 column. Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation. (3089)

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J. Cell Biol. 279, 50069–50077. p51/p63 controls subunit 3 of the major epidermis integrin anchoring the stem cells to the niche. 2004

Kurata, S., Okuyama, T., Osada, M., Watanabe, T., Tomimori, Y., Sato, S., Iwai, A., Tsuji, T., Ikawa, Y. and Katoh, I.

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3.  The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit.    (3225)

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Plant Physiol. 126, 1416–1429. Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional CaaL box motif and a proximal polybasic domain. 2004

Caldelari, D., Sternberg, H., Rodríguez-Concepción, M., Gruissem, W. and Yalovsky, S.

Notes: Using the SV Total RNA Isolation System before RT-PCR, expression levels of AtGGT-IB were compared in flowers, leaves, stems, and root tissues of wildtype and era1-2 Arabidopsis plants. First-strand synthesis was performed with M-MLV Reverse Transcriptase using 500ng of RNA and an equal amount of oligo(dT). A 1:10 dilution of this product was used for amplification. (2855)

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