Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes

Expression vectors for cDNA of the κ and λ1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. κ and γ1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive κ and γ1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of y 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of κ cDNA under the control of the S V40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) κ determinant, including introns. Such an entire κ gene led to expression of the light chain at levels double those with the κ cDNA construction using the SV40 promoter and about 35 times as high when using κ cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

Use of an electrostatic sprayer for control of anopheline mosquitoes

The Electrodyn sprayer was compared with a compression sprayer (Hudson X-pert) for residual application of cypermethrin, a pyrethroid insecticide, to control the malaria vectors Anopheles arabiensis Patton and An. funestus Giles in experimental huts at Magugu in Tanzania. The time taken for hut spraying, 2-2.5 min per hut, was similar for both types of sprayer. Two or three huts were treated internally with cypermethrin at 40 or 80 mg a.i./m2 using Electrodyn formulation for comparison with 80 mg a.i./m2 using wettable powder formulation. Each of the twelve huts (including five untreated controls) was fitted with window exit traps and either louvre or verandah traps for mosquito sampling. The Electrodyn sprayer was fitted with a pair of elbowed deflectrodes to direct the positively charged spray droplets onto walls and ceiling. All treatments gave 94-100% mortality-rates of indoor-resting anophelines throughout the evaluation period of 11 weeks post-spray. Reductions of An.arabiensis and An.funestus females by 10-42% and 62-91%, respectively, in rooms and by 72% and 51% in exit traps indicated that cypermethrin deterred mosquitoes from entering the huts. Overall mortality-rates of mosquitoes were 66% of both species in huts treated with 40 mg/m2 Electrodyn, 43% An.funestus and 71% An.arabiensis due to 80 mg/m2 Electrodyn formulation and 49% An.funestus and 64% An.arabiensis due to 80 mg/m2 WP formulation (no significant differences). It is concluded that the Electrodyn sprayer with deflectrodes is a convenient and effective means of residual house-spraying with pyrethroid insecticide for malaria vector control.     

DNA probes to identify members of the Anopheles farauti complex

DNA probes have been constructed to distinguish between the members of the Anopheles farauti complex of mosquitoes known as species numbers 1, 2 and 3. Partial genomic libraries of the three known species were exposed to labelled total genomic DNA from each species. Colonies showing differential hybridization were selected for further testing. These probes were found which allow identification of the three known species: probe pAf1 (160 bp fragment) hybridizes to DNA from An. farauti nos. 1 and 2; probe pAf2 (95 bp fragment) hybridizes to DNA from An. farauti no. 2 only; and probe pAf3 (1.3 kb fragment) hybridizes strongly to DNA from An. farauti no. 3, less to no. 1 and faintly to no. 2. Increasing the stringency of hybridization reduced the cross-hybridization of probes pAf1 and pAf3. Only radioactively labelled probes were tested. Males and females and individuals from diverse habitats and localities showed the same species/probe hybridization characteristics. This technique allows faster identification of the sibling species than previous methods, and has the added advantage that it allows air-dried and alcohol stored specimens to be identified.     

Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin

Summary Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5 end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.     

Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.