Egg Whites Aren't Just for Chicks (or Dieters)

It has recently become increasingly clear that three-dimensional (3-D) cell culture systems recapitulate the structural and physiological environment of cells in vivo better than traditional two-dimensional monolayer cell cultures. Commercially available 3-D cell culture media contain a reconstituted basement membrane preparation of extracellular matrix proteins derived from a mouse sarcoma cell line. Due to the expense of these commercial 3-D cell culture media, B. Kaipparettu and colleagues in the lab of S. Oesterreich at Baylor College of Medicine (Houston, TX) have developed a cost-effective and reliable alternative simply using avian egg white. To test the ability of egg white to support 3-D cell culture, they used the MCF10A immortalized human breast epithelial cell line, whose growth in 3-D media is well characterized. They demonstrated that MCF10A cells grown in egg white formed characteristic acini—spheres consisting of polarized epithelial cells surrounding a lumen—with the same morphology, apicobasal polarity, pattern of apoptosis, and growth rate as those grown in reconstituted basement membrane medium. Five established human cell lines derived from different tissues as well as transformed mouse mammary epithelial cells were tested and each showed comparable 3-D morphologies in both media, verifying that the ability of cells to grow in egg white is neither tissue- nor species-specific.

(See “Novel egg white–based 3-D cell culture system” on page 165.)

Pin the Cell: mRNA Isolation with Microneedles

For researchers interested in studying mRNA expression patterns by directly sampling mRNA from living cells or tissues at a high temporal and spatial resolution, the typical methods for the small-scale isolation of mRNA from cells and tissues are unsuitable since they are destructive and can result in mRNA that is contaminated with DNA. In this issue, P. Scherp and K. Hasenstein at the University of Louisiana at Lafayette (Lafayette, LA) describe their “solid phase gene extraction” (SPGE) technique to rapidly and nondestructively sample and isolate mRNA from live materials using glass microneedles coated with oligo-dT or gene-specific oligonucleotide sequences. The mRNAs that are directly hybridized to the probe can be extracted in sufficient quantities to be converted into cDNA and analyzed by real-time PCR without the need for pre-amplification. Since the use of the glass microneedle is generally nondestructive, depending on the target cell or tissue, this allows for repeated sampling at different locations or times. The authors validated their SPGE method by using it to observe the well-characterized distribution of bicoid and nanos mRNA, which are reciprocally localized along the anterior-posterior axis of the Drosophila egg. They also demonstrated its applicability for the study of actin mRNA distribution in germinating flax seeds.

(See “Solid phase gene extraction isolates mRNA at high spatial and temporal resolution” on page 172.)

Plugging the Leak: Basal Expression from Inducible Transgenes

Inducible transgene expression in mammalian cells is an important tool for the analysis of gene function. A critical necessity for these systems is the tight control of basal transgene expression, both to reduce the potentially harmful effects of expression in uninduced cells and to allow for the clear discrimination of biologically relevant effects due to induced gene expression. While tetracycline (Tet)-regulated inducible transgene systems are the most commonly used and have advantages over other systems, they can suffer from leakiness in the level of basal transgene expression. The various remedies attempting to reduce or eliminate this problem have had mixed success. D. Pham and colleagues working in the lab of S. Pitson at the U. of Adelaide (Adelaide, Australia) have developed a novel method to better suppress this leakiness by incorporating AU-rich mRNA destabilizing elements (ARE) into the 3′ UTR of inducible constructs. They demonstrated that this modification could eliminate the relatively high basal leakiness of sphingosine kinase 1 (SK1) expression from a Tet-inducible vector with only a slight decrease in the maximum level of fully induced SK1 as compared to that from the construct without the AREs. This approach should also be applicable to any other inducible protein expression system with leaky basal expression.

(See “Attenuation of leakiness in doxycycline-inducible expression via incorporation of 3′ AU-rich mRNA destabilizing elements” on page 155.)