Lentiviral Vectors Find Stability in E. coli

Generally, lentiviral expression vectors contain large, directly repeated DNA sequences from the long terminal repeats (LTRs) of the retroviruses from which they originated. Obtaining the desired clones of these vectors in typical Escherichia coli hosts (for example, DH5α or TOP10) is particularly difficult. Deletion of the regions between the LTRs removes most of the important lentiviral sequences and produces a small plasmid with only the antibiotic resistance marker and replication origin, resulting in recombinant plasmids that are strongly selected for. In this issue, C.S. Chakiath and D. Esposito at SAIC-Frederick (Frederick, MD) show that the recently developed reduced-genome E. coli strain MDS42 (and its derivative MDS42recA) not only increases the stability of lentiviral expression constructs but is also compatible for use in Gateway® recombinational cloning. Unlike the strain STBL3, which was developed to overcome these recombination problems but is slow growing, sensitive to phage T1, and difficult to make competent for transformation, MDS42 suffers none of these defects while reducing plasmid recombination events, possibly because of the loss of insertion sequence (IS) elements. At 1 × 10⁸ cfu/µg, the transformation efficiencies for MDS42 and MDS42recA were at least two times better than those for STBL3 and the cells grew faster. Using both reduced-genome strains, STBL3, DH5α, and TOP10, the scientists tested for plasmid stability of a lentiviral expression clone generated by a Gateway LR recombination reaction. In terms of the number of colonies with the correctly sized clone obtained at 30° and 37°C, MDS42 is clearly superior to DH5α and TOP10 and is the same or better than STBL3. LTR recombination is even more undesirable in multisite Gateway recombination, which demands an E. coli strain with higher transformation efficiency and yields fewer colonies for each reaction. MDS42 repeatedly produced colonies containing the expected size DNA and had higher transformation rates than STBL3. MDS42 and MDS42recA reduce the number of DNA constructs that need to be prepared for cloning, greatly improving the efficiency of high-throughput cloning.

(See “Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli” on page 466.)

Gels and Cells: 3-D Microtissue Formation

The self-assembly of tissue-culture cells into 3-D bodies in vitro has been increasingly useful for examining fundamental questions in cell, developmental, and cancer biology. Since 3-D microtissue structures more closely approximate the topology of cells in tissues and organs than do conventional 2-D cell culture systems, they more likely replicate the cell-to-cell interactions, both direct and indirect, that govern basic biological processes occurring in vivo. Among the techniques for forming microtissues, simpler methods involving the self-assembly of cells using hanging drops or spinner cultures suffer several handicaps: they produce microtissues formed only in spheres in a high-shear environment at low throughput. More sophisticated technology such as photolithography, laser tweezers, and cell printing can overcome these limitations but are expensive or difficult to implement. Napolitano and colleagues at Brown University (Providence, RI) have devised a versatile and cost-efficient method using micromolded, nonadhesive agarose hydrogels for the production of 3-D microtissues with simple or complex forms and comprised of uniform or mixed cell populations. The hydrogels are cast with reusuable and autoclavable polydimethylsiloxane micromolds that form multiple recesses of the desired shape into the hydrogels, which are designed to fit into 6-, 12-, or 24-well plates. Suspended tissue-culture cells placed over the hydrogels settle into the recesses. Because the cells do not adhere to the hydrogel, they spontaneously self-assemble through cell-cell binding into shapes that conform to the recesses, which can vary from simple (spheroids, rods, toroids) to complex (loop-ended dogbones, honeycomb lattices). Tumor spheroids can be grown from the clonal expansion of single cells, with their proliferation conveniently monitored via the WST-1 assay. Mixtures of two or three differentially fluorescent-labeled cell lines were used to generate heterotypic multilayered structures, and cell sorting could be easily observed by confocal microscopy. Given its versatility and scalability, as well as its compatibility with standard cell culture reagents and microscopy methods, these micromolded agarose hydrogels should greatly expand the application of 3-D microtissues in various fields, including tissue engineering and high-throughput drug screening.

(See “Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels” on page 494.)

Undisguised FRET: De-Quenching Assay Quantifies Biotin Conjugates

The biotin-avidin interaction, with its high specificity and strong binding affinity, is the lynchpin for a wide variety of biological detection systems. The performance of many of these systems depends upon the number of biotins attached to each macromolecule of protein or nucleic acid. While assays have been developed to detect and quantify biotin directly, the existing alternatives lack at least one of the following four desirable characteristics: no-wash, high-throughput, rapid readout, and sensitivity. To alleviate these shortcomings, Batchelor and coworkers at Invitrogen (Eugene, OR) have developed a high-throughput method for determining covalently bound biotin based on the FRET system. This assay uses a complex consisting of Alexa Fluor® 488 dye-labeled avidin and a fluorescence emission quencher dye, HABA (occupying the biotin binding sites of the avidin). Biotin displaces HABA from the complex as it binds to the dye-labeled avidin. Removing the quencher increases fluorescence intensity in direct proportion to the amount of biotin present in the sample. Biotinylated macromolecules can be assayed either intact to determine the effective (i.e., non-sterically hindered) biotin amount or enzymatically digested for the total biotin amount. The authors show that a very low level (4 pmol) of biotin in a 0.1-mL volume is detectable within 15 min after adding the sample to the reagent. The assay is amenable to high-throughput platforms, is highly sensitive, has a rapid readout, and requires no washing step.

(See Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids” on page 503.)

Proximity Ligation Assays Faster, Cheaper, Easier

Proximity ligation assay (PLA) is a method for sensitively quantifying proteins, whereby the binding of affinity probes to a target protein results in the formation of a DNA molecule that can be measured by PCR. The most recent and sensitive version, the triple-binder proximity ligation assay (3PLA), employs three different proximity probes, each consisting of a unique oligonucleotide attached to antibodies against the same protein. Simultaneous binding of these three proximity probes to their target via the antibody portion brings the attached oligonucleotides into close proximity, and a subsequent ligation reaction generates a longer DNA sequence that be quantified by real-time PCR. Typically, proximity probes are prepared by noncovalent attachment of a biotinylated antibody to a single-stranded oligonucleotide covalently linked to streptavidin through maleimide conjugation. This biotin-streptavidin (B-STV) approach allows the same set of streptavidin-oligonucleotides to be used with any biotinylated antibody to produce proximity probes against a wide range of target proteins. In situations where a larger set of different oligonucleotides may be desirable, however, this approach would be time-consuming and tedious, due to the maleimide conjugation and purification of the many streptavidin-oligonucleotide molecules involved. To circumvent this difficulty, the developers of the original proximity ligation assay and 3PLA, Edith Schallmeiner, Ulf Landgren, and colleagues at Sweden's Uppsala University have devised an alternate protocol for faster, lower cost, and more convenient proximity probe preparation. Instead of covalently attaching an oligonucleotide to the streptavidin, the new method allows a biotinylated oligonucleotide to bind to streptavidin first, followed by binding of the biotinylated antibody. Proximity probes generated by this biotin-streptavidin-biotin approach (B-STV-B) were demonstrated to be comparable to those from the B-STV method for detecting vascular endothelial growth factor diluted in either buffer or 1% or 10% human plasma. The Uppsala team has also increased the convenience of the PLA assay by separating the real-time PCR step from the preceding affinity probe binding and ligation steps, allowing for the processing of large sample sets when real-time PCR capacity is limited.

(Click to enlarge)

(See “Self-assembly of proximity probes for flexible and modular proximity ligation assays” on page 443.)