Generating Knockouts of somatic cells of metazoans is filled with difficulty. Although the chicken DT40 cell line has been used with considerable success, the functional information derived from this model system may not be directly applicable to investigation human gene function. Recently, a group of researchers based in Japan reported that a human pre-B cell line known as Nalm-6 enables highly efficient gene targeting. Despite this advance, the process of constructing the appropriate vectors can be time-consuming and frustrating. Looking to ameliorate this situation, liizumi et al. describe a streamlined protocol for construction and use of gene-targeting vectors. The strategy uses recombinational cloning and employs positive and negarive selection markers. By following the author's clearly presented protocol, researchers interested in homozygous knockout of a human gene can expect to derive the desired cell line in 2-3 months.-Page 311
On Display
Phage display is typically thought of as a means to select novel peptide sequences capable of binding with high affinity to a factor of interest. However, the system is also highly appropriate for uncovering the bindings partners of uncharecterized ORFs. In this latter approach, fragments from a cDNA library are expressed in-frame with one of the phage coat proteins. Clearly, the efficiency of ORF selection is compromised by fragments cloned in reverse orientation or containing a stop codon before the coat protein coding sequence. Therefore, Hust et al. turned to a helper phage that does not express the coat protein in order to eliminate the production of any phage that does not include sequences encoding a viable ORFcoat protein fusion. As expected, the replacement of traditional helper phage M13K07 with Hyperphage (which has a deleted, nonfunctional minor coat protein) significantly enriches for phage bearing in-frame ORF sequences. Significantly the method described by Hust et al. does not require a subcloning step–unlike other enrichment strategies. The authors expect their Hyperphage-based approach to be of particular value for vaccine development and functional genomics. -Page 335
Electron and light microscopy can be considered complementary techniques, but the sample mounting procedures are vastly different, and cells prepared for one application are useless for the other. Recently however, a commercial product that allows SEM of a fully hydrated sample has become available. This advance has significantly simplified the EM sample preparation process and allows the same samples to be viewed using either light or electron microscopy. Unfortunately, the fluorescent stains that are typically used in light microscopy do not provide adequete contrast for meaningful EM imaging. To address this limitation, Timp et al. investigated whether quantum dots could do double duty as both an electrondence compound and a fluorescent stain. Although cells incubated with quantum dots did not show the resolution or signal intensity obtainable with traditional EM contract agents such as uranyl acetate or colloidal gold, substantial cellular detail is nevertheless observable. And, uniquely, the quantum dotstained cells can be used directly in fluorescence microscopy, thereby permitting correlative analysis by sequential light and electron microscopy. This simple technique should be strongly suited to situations in which cells positive for a particular marker can be identified quickly by fluorescence imaging, and subsequently analyzed in detail by EM. -Page 295
SPA Treatment for p53
The scintillation proximity assay (SPA) is just one of many techniques that are used to detect the binding of proteins–particularly transcription factors–to specific DNA sequences. The Principle behind the technology is that the two main components of the system, a scintillation bead and a radiolabeled ligand, create a visible light signal when brought together in close proximity. The SPA, available as a commercial kit, is simple to set up and adaptable to many different as say applications. In this issue, Gal et al. modify this assay for application as a rapid screening tool for studying the binding of p53 to 3H-labeled DNA in a high volume 96-well format. p53 is an obvious target since its role in cancer progression is well-known, and it is one of the most frequently mutated genes found in neoplasias. The authors demonstrate that the assay is sensitive (>0.5 ng p53 detectable), rapid, specific, quantifiable, and high-throughput. They were also able to examine and visualize, in a manner not easily done with other assays like gel shift analysis, the dissociation kinetics of DNA from p53. -Page 303
Do Ask, Do Tell
Since its sequencing in the mid 1980s, the use of the internal ribosome entry site (IRES) motif has become increasingly popular as a means to achieve enhanced translation efficiency in monocistronic vectors, as well as the simultaneous expression of two genes by using a bicistronic setup. Some aberrant behaviour with commercialized versions of these vectors has been described, such as, in the case of the latter vector, differential expression depending on the order of the genes. However, Dr. Ann Palmenberg and her group, who have published extensively on IRES, caution that there are additional reasons to be concerned. During initial commercial- ization in the early 1990s, an error introduced an extra base pair in a run of adenines to create A7. This was cor- rected in a later version of the vector (returning it to A6), but both the A6 and A7 forms of the IRES sequence have been used in the multitude of customized vectors produced in different laboratories over the years with a certain amount of disregard for their origin. This is troubling when one considers that the two sequences behave differently and use some dissimilar translation factors. In this paper, Bochkov and Palmenberg examine how the origin of IRES elements may affect expression in bicistronic systems and conclude that caution should be exer- cised in IRES selection and placement within expression vectors. -Page 283
Playing Tag with Lectins
Since the development of the first expression vectors, the ability to isolate pure recombinant protein cleanly has been top of many researchers’ wish lists. Naturally occurring molecular affinities, such as that between avidin and biotin, or between hexahistidine moieties and elemental nickel, have been hijacked for this cause. Another high-affinity system with attractive properties is the binding of plant lectins to a variety of carbohydrate molecules, and it is just such a system described by Tielker and colleagues in this issue. A novel lectin tag is described, consisting of a D-mannose-binding LecB lectin from Pseudomonas aeruginosa fused to a protein of interest via a cleavable enterokinase linker, which allows one-step purification. Affinity chromatography over a mannose agarose matrix of a YFP-LecB fusion protein was demonstrated with elution in a tight peak using D-mannose. Enterokinase-driven cleavage could then be carried out to remove the unwanted tag, and the purified protein obtained following a second pass over a mannose column, followed by a single wash step. LecB does not appear to effect the functionality of the protein to which it is fused and is also highly temperature stable, potentially making it a candidate for purification of proteins from thermophilic organisms. -Page 327
