Plotting Foundations for Tissue Engineering

Cell-based therapies may be within our grasp, but only if researchers can develop substrates to help select and grow complex functional tissues in vitro. To reach this goal, developers must be able to predict how changes in substrate properties—chemical, mechanical, and topological—will affect cell attachment, survival, migration, growth, differentiation, and a host of other traits. And this, in turn, demands iterative rounds of combinatorial biomaterial synthesis; rapid, quantitative assays of cellular responses to changes in substrate composition, and computational models that make sense of it all. Assays for cellular responses to biomaterials have so far been largely subjective—and static and slow, dependent on fixing tissue samples for individual examination. In this issue, M.D. Treiser, P.V. Moghe, and coworkers at Rutgers University (Piscataway, NJ) describe high-resolution, high-throughput imaging of cell-based fluororeporters to quantify metrics of cytoskeletal features and cell functional endpoints (such as cell growth, cell adhesion, and cell functional endpoints (such as cell growth, cell adhesion, and cell attachment strength). Working on selected mammalian cell lines engineered with a series of green fluorescent protein (GFP) fusion genes, the researchers were able to image live cells on a series of green fluorescent protein (GFP) fusion genes, the researchers were able to image live cells on a series of biomaterials from a combinatorial library synthesized from varying proportions of desaminotyrosyl tyrosine alkyl ester, desaminotyrosyl tyrosine with a free acid, and polyethylene glycol (PEG). The Rutgers group found that high-content imaging yielded many quantifiable descriptors of cell cytoskeletal and other morphometric data. The data show that the fluororeporters reveal changes in cell behavior in response to substrate hydrophobicity, stiffness, charge, and protein-repulsive character (increased PEG levels lower protein adsorption). Ideally, quantifying cell responses will lead to models that predict how specific biomaterials will affect cellular development. Ultimately, perhaps, the method will lay the foundation for a general approach to producing cell populations with predefined characteristics.

(See “Profiling cell-biomaterial interactions via cell-based fluororeporter imaging” on page 361.)

Four-Part Harmony, Jazzy Fusion

Generally, DNA constructs are joined by ligation at restriction enzyme sites, and construct design is limited by the availability of unique sites in the linearized vector and gene. In this issue, G.J. Freeman and coworkers at the Dana-Farber Cancer Institute (Boston, MA) report on their advancements in creating fusion proteins, modular vectors, and new mutagenesis strategies using Clontech's In-Fusion™ system. The system can join any two pieces of linear DNA that end in 15 identical base pairs (which can be engineered by PCR) through a ligation-independent reaction. The reaction appears to use poxvirus DNA polymerase's unique 3′-5′ exo-nuclease activity to create 5′ overhangs that can then anneal to each other. Transformation into E. coli will repair any nicks or gaps in the resulting joined, circular plasmid DNA. This system was originally reported for the two-piece joining of an insert with a restriction enzyme-digested plasmid. In this month's BioTechniques report, the researchers demonstrate that In-Fusion has the ability to join as many as four DNA fragments in just one reaction, without adding unwanted amino acids in the case of fusion protein constructs. They success-fully designed a CD101-IgG3 fusion protein by joining four pieces of DNA encoding a secretory signal, a CD101 extracellular domain, and an IgG3 Fc domain with an expression vector. More generally, the DNA segments can be joined as desired to create modular vectors with separate components that can be removed and replaced. The Dana-Farber researchers devised what they termed In-Fusion replacement to replace any segment of plasmid DNA with another sequence (the replacement segment) by either of two methods: joining two PCR boundary fragments with the replacement segment and plasmid digested with two unique restriction enzymes; or joining a PCR-amplified linear plasmid with the replacement segment DNAs. The authors also used In-Fusion to facilitate DNA mutagenesis in two ways. (i) Two adjacent DNA segments are PCR amplified using primers incorporating the desired mutation and joined with restriction enzyme-digested plasmid, thus incorporating the mutation. (ii) Or mutagenic sense and antisense primers are used to PCR-amplify the circular plasmid, producing a linear DNA strand that is then recircularized via an In-Fusion reaction. Using these various strategies, multipiece In-Fusion joining is powerful methodology to create seamless fusion constructs and to generate any desired alteration in a DNA plasmid sequence.

(See “In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations” on page 354.)

Skin-Deep on Zebrafish

Zebrafish is a very useful model for studying vertebrate organogenesis and genetics, boasting such features as the rapid growth and transparency of the embryo during early development. While a number of genes controlling development of various zebrafish organs have been identified from mutants isolated by genetic screens, very few epidermis mutants have been obtained, hampering an understanding of zebrafish skin development. Epidermal studies in zebrafish larvae are complicated: while the epidermis is very thin and formed by flat layers of cells, the larva's round shape makes it difficult to obtain horizontal sections for histological and electron microscopic analysis. Existing techniques are not suitable for large-scale epidermal mutagenesis screens. In this issue, Villava and colleagues at Universidad Nacional Autnoma de Mxico have developed a simple and fast freeze-crack procedure for recovering epidermal cells of the early larva attached to a glass slide. The authors show that the captured epidermis is useful for studying subcellular structures such as cell membrane, nuclei, and the Golgi apparatus by labeling with fluorescent markers, followed by analysis using differential interference contrast (DIC) microscopy. They demonstrated that the epidermis from multiple larvae (15–75) can be easily prepared and analyzed in about 2–4 h. The freeze-crack method appears to have potential as a tool for epidermal studies and genetic screening, among other applications.

(See “Freeze-crack technique to study epidermal development in zebrafish using differential interference contrast microscopy and fluorescent markers” on page 313.)

TAPping Mammalian Protein Complexes

Native affinity purification of tagged target proteins bound to their interacting partners is a key tool of proteomics. To improve the technique's specificity, researchers developed the tandem affinity purification (TAP) tag, consisting of two different peptide tags arranged in tandem and separated by a specific protease recognition site. Fused to the bait (target) protein, the TAP tag permits an initial affinity purification targeting the outer tag. Protease cleavage releases the concentrated protein-prey complexes, which are then re-isolated via a second affinity purification based on the inner tag and identified via mass spectrometry. This three-step process increases specificity and yields of the purified interacting proteins (prey) of target proteins. The system works well in yeast, for which it was originally developed, but has generated inconsistent results when adapted to mammalian systems, sometimes producing relatively low yields of bait proteins and co-purified prey. To broaden the variety of available TAP tools, Yisong Wang and colleagues at the Oak Ridge National Laboratory (Oak Ridge, TN) have constructed a series of five novel dual-tag purification vectors. Each possesses a different set of tandem affinity tags, some located at the amino-terminal end and others at the carboxy terminus. Further improvements include an additional protease site to improve cleavage efficiency and a tetracysteine motif (CCPGCC) that binds the conditionally fluorescent Lumio™ compound, allowing visualization of the fusion protein in live cells and in gel-electrophoresed cell lysates. The fusion protein can be expressed under the control of constitutive or tetracycline-regulatable promoters (the latter allows conditional expression of proteins that might otherwise be toxic to cells). With their combination of additional tandem affinity tags and various improvements, these versatile constructs should greatly increase the likelihood that a target protein and its interacting partners can be isolated in sufficient quantities to permit identification by mass spectroscopy.

(Click to enlarge)

(See “Dual-tagging system for the affinity purification of mammalian protein complexes” on page 296.)