A genetically engineered cell culture releases RNA into the medium, allowing its activity to be measured repeatedly

A genetically engineered cell culture releases RNA into the medium, allowing its activity to be measured repeatedly

On September 1, Cell published a study describing a method for repeatedly measuring gene activity in the same living cell culture. The authors introduced a genetic construct into the cells, causing them to package some of their RNA into virus-like particles and release it into the culture medium. Successive samples of the medium can then track changes in gene activity while leaving the culture intact for further measurements.

The RNA in a cell indicates which genes are active. The complete set of these RNA molecules is called the transcriptome. Conventional sequencing, which determines RNA sequences, destroys the cells, so a time series must be assembled from separate samples. The authors tested whether they could obtain such a series from the same living culture.

They introduced the MMLV Gag gene, which encodes a structural protein of the murine leukemia retrovirus, into the cells. Near the cell membrane, this protein assembles virus-like particles that capture RNA from the cytoplasm and bud into the medium. The control condition used a truncated form of Gag that cannot form these particles, and substantially less RNA was detected in the medium. After 48 hours, the lysed cell contents contained RNA from 12 931 genes, and RNA from 11 598 of those genes was also found in the medium. Their activity levels agreed well, with a correlation coefficient, a measure of similarity, of 0.797. Each medium sample captures the RNA signal accumulated during the interval between collections, primarily from the cytoplasm.

The researchers then tested the method during a four-day differentiation of stem cells into neuron-like cells. A comparison of day 0 and day 4 in the lysed cell contents identified 772 genes whose activity had changed. Serial samples of the medium showed the same changes. This allowed the authors to track daily changes within a single culture.

The authors also applied the method in a microfluidic device, a small system of channels through which culture medium flows. Inside the device, endothelial cells, which line blood vessels, formed a vascular network in a gel. The endothelial cells were cultured either with lung fibroblasts, a type of connective tissue cell, or with endometrial stromal cells, which belong to the supporting tissue of the uterine lining. Each condition used three devices. For seven days, the researchers collected medium daily from the same six devices and observed how the cellular environment produced different changes over time in gene expression programs associated with blood vessel formation.

This seven-day series required six devices. A destructive measurement would have required 42 independent devices.