Notes: The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjunct cancer therapy drug used for CD20-positive B-cell lymphoma. CD20 PCR products amplified from genomic DNA were cloned into the pTARGET™ Mammalian Expression Vector. These CD20 mutant constructs were stably introduced into K562 chronic myelogenous leukemia cells by electroporation and selected using G-418. One microgram of CD20 mutant construct DNA was transcribed and translated using an in vitro translation kit from Promega. (4032)

Notes: This study examined the expression levels of each UGT isoform in human liver and evaluated the variability between individuals. Total RNA from appropriate human tissues or various cell lines was used for RT-PCR of various human UDP-glucuronosyltransferases (UGT) cDNAs. The amplimers were cloned into the pTARGET™ Mammalian Expression Vector and verified by sequencing. The UGT vectors were linearized by restriction enzyme digestion and used for standards in real-time RT-PCR analysis. (4034)

Notes: The authors used a systems biology approach to examine the transcriptional regulation of water channel aquaporin-2 (AQP2). A 1,511bp fragment from the 5´-flanking region of the mouse AQP2 gene was amplified from mouse tail DNA and cloned into the pGEM^®-T Vector. This construct was then digested with two restriction enzymes and cloned into a double-digested pGL3-Basic Vector. Full length Elf3, Elf5 and Ehf cDNA, members of the ETS family of transcriptional regulators, were amplified, sequenced and ligated into the pTARGET™ Mammalian Expression Vector. LLCPK1 cells were cotransfected with AQP2-pGL3 reporter and one of the pTARGET™ constructs. Reporter activity was measured using 20µl of cell lysate in a luciferase assay. (4033)

Notes: To examine the role of the membrane form of macrophage colony–stimulating factor(mM-CSF) in the hematopoietic system, RT-PCR was used amplify the cDNA of human mM-CSF from J6-1 cells, a human leukemia cell line. The PCR product was digested and cloned into the pTargeT™ Mammalian Expression Vector. After sequencing to verify the sequence, the construct and empty pTargeT™ Mammalian Expression Vector were purified and used to transfect Namalwa and Ramos cells, human Burkitt’s lymphoma cell lines, in 24-well plates. The transfected cells were then selected for stable expression of the transfected vector using 1.4 mg/ml G418. Expression of mM-CSF and neomycin (in the empty vector) was confirmed using RT-PCR. These cells were injected into mice and the oncogenicity of the cells determined using antibody staining of tissues. (3989)

Notes: To examine the causes of Factor V (FV) deficiency, the authors examined transcript splicing and its mutated variations. Three regions of human FV (F5) were amplified from a healthy individual and the PCR products cloned into the pTargeT™ Mammalian Expression Vector. Three identified mutations from people with FV deficiency were introduced by site-directed mutatgenesis. All constructs were sequenced before transfection into HeLa cells. After 48 hours, the total RNA was purified and the splicing pattern of the wild type and mutant constructs were analyzed by RT-PCR. The mutant constructs were also transfected into HepG2 cells and tested for nonsense-mediated mRNA decay (NMD) with or without NMD inhibitors (puromycin, cycloheximide, and wortmannin) using RT-PCR. (3992)

Notes: Sulfatide is present in mammalian organs where influenza A replicates. Ceramide galactosyltransferase (CGT) and cerebroside (galactosylceramide) sulfotransferase (CST), which synthesize sulfatide, were cloned by PCR into the pTargeT™ Mammalian Expression Vector and the pGEM®-T Easy Vector, (CST with or without a three base insertion), respectively. The two genes were removed by restriction digestion and cloned into pIRES-neo to forma bicistronic construct. Arylsulfatase A (ASA), which degrades sulfatide was also amplified and cloned into the pGEM®-T Easy Vector, before being subcloned into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418. (3990)

Notes: The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase^® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times. (3691)

Notes: This paper explored the process by which the Eph receptor EphA2 regulates repulsive response in prostatic carcinoma cells via the ephrinA1 ligand. Focal adhesion kinase (FAK) non-related kinase (FRNK; a target in the ephrinA1 ligand cascade) was subcloned into the pTargeT™ Mammalian Expression Vector. This mutant FAK was transfected into PC-3 cells and subjected to a GST-pulldown assay to examine RhoA activation. In addition, PC-3 cells transfected with several targets in the EphA2/ephrinA1 activation cascade including FRNK were subjected to an in vitro three-dimensional migration assay. (3690)

Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

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)

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

Notes: To test the effect that Xenopus laevis UDP-glucose dehydrogenase (xUGDH) expression has in mammalian cells, the xUGDH ORF was amplified, purified, A-tailed and cloned into the pTARGET™ Mammalian Expression Vector. Two clones were selected: one in the sense orientation and one in the antisense orientation. The constructs were confirmed by DNA sequencing and expression in a TNT^® T7 in vitro transcription/translation system. Five micrograms of the plasmids were transfected into AoSMCs and human aortic smooth muscle cells, and UGDH activities werw measured 48 hours post-transfection. (3497)

Notes: The cDNA for the IL-1F7b protein was amplified from a human spleen cDNA library and directly subcloned in the pGEM^®- T Easy Vector and sequenced. The clone was transferred to a bacterial expression vector with a purification tag. The expressed protein was used to make a polyclonal rabbit antibody and antibody purification column. The cDNA for the IL-1F7b was reamplified and directly subcloned into the pTARGET™ Mammalian Expression Vector. The cDNA was stably expressed in RAW264.7 mouse monocyte/macrophage cell line.  Lysates from the cells stably expressing IL-1F7b were used to test the antibodies by western blotting. Expressing cells were also tested by immunocytochemistry. (2598)

Notes: The cDNA for the TERE1 gene was amplified by PCR and directly subcloned into the pTARGET™ Mammalian Expression Vector. The 338-amino acid (36.8kDa) TERE1 protein was stably expressed in three human bladder transitional cell carcinomas (J82, T24 and 1376 TCC) and the human embryonic kidney cell line, HEK 293. Stable transfectants were selected with the antibiotic G-418. Overexpression of the protein caused 80-90% inhibition of cellular proliferation in two of the transitional cell carcinoma cell lines and inhibition of aneuploidy in the third. Transfections were accomplished in 24-well plates with the Tfx™-20 Transfection Reagent. Excellent details of the 1 hour transfection protocol are presented. (2596)

Notes: The cDNA for the short isoform of the caspase-2 mRNA was amplified and cloned directly into the pTARGET™ Mammalian Expression Vector. The protein was stably expressed in U937 human leukemic cells by selection with the antibiotic G-418.  Stable clones were identified by PCR of the genomic DNA of selected colonies with primers to the T7 promoter of the pTARGET™ Vector and the downstream primer of the caspase-2 message. Overexpression of the protein interfered with etoposide-mediated apoptosis. (2597)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express a portion of the ARA9, a protein that interacts with the aryl hydrocarbon receptor. The expressed protein was used in a two-hybrid assay using a GAL4-fusion protein containing a portion of the aryl hydrocarbon receptor. Interaction of the two proteins is reported by activation of the luciferase in the pG5-luc Vector, which is a portion of the CheckMate™ Mammalian Two-Hybrid System. The two-hybrid studies were performed in COS-1 cells and were controlled with a cotransfection with a β-galactosidase vector. Cells were lysed with the Passive Lysis Buffer and luciferase activities determined with the Luciferase Assay System. (0842)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express the TRAIL-3 and the TRAIL-4 receptors. The proteins were expressed in primary human melanoma cells. (0098)

Notes: Three proteins were amplified with an HA tag and cloned into the pTargeT™ Mammalian Expression Vector for expression in transfected cells. The proteins were expressed in COS-7 cells, and were used to study protein association in cell extracts and in intact cells. (2153)

Notes: The pTargeT™ Mammalian Expression Vector System was used to clone and express the human cytosolic 5'-nucleotidase II, a 65kDa protein. The protein was expressed and assayed in COS-7 monkey kidney cells and H9c2 rat myoblast cells. The pSV-beta Galactosidase Control Vector was used as a control for transfection efficiency. (0431)

Notes: The XRCC3 gene was amplified from a human testis cDNA library and directly subcloned into the pTARGET™ Mammalian Expression Vector. The insert was sequenced, then electroporated into the irs1SF hamster radiation-sensitive cell line. Stable transfectants were isolated by G-418 selection and used in chromosome segregation studies. (2599)

Notes: The T-cell receptor of interest was amplified and subcloned in the pTARGET™ Mammalian Expression Vector. Large amounts of the vector were purified with the Wizard^® Plus MegaPreps DNA Purification System and used as a DNA vaccine in Lewis rats. (0701)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express MIG (monokine-induced by Interferon-gamma) and IP-10 (interferon-inducible protein 10) in A549 human adenocarcinoma cells. Stable transfectants were obtained through G418 selection. (2058)

Notes: Subunits of the cDNA for methionine adenosyltransferase were amplified from a plasmid using Taq DNA Polymerase and directly cloned into the pTARGET™ Mammalian Expression T-Vector. The sequence of the cloned inserts were confirmed with the fmol^® DNA Cycle Sequencing System. The constructs were transiently transfected into COS-1 cells with the TransFast™ Reagent at a 1:1 ratio. Excellent detail is provided for the transfection. (1094)

Notes: The 873 amino acid kinase repressor of Ras (KSR-1) was amplified to add a flag tag and the resultant product was cloned into the pTARGET™ Mammalian Expression Vector. The protein was expressed in COS7 cells and used for subcellular fractionation studies. (1434)