Functional Characterization of Insulation Effect for Synthetic Gene Circuits in Mammalian Cells.

Insulators are noncoding gene regulatory elements in eukaryotic genome, which function as chromatin partitioning boundaries, and block interference across different chromatin domains. To facilitate modular construction of synthetic gene circuit that is usually composed of multiple transcription cassettes, unwanted cross-regulations between different cassettes should be avoided. Here, we developed a quantitative method to characterize the functional effect of three insulators on the cross-regulations of six promoters in mammalian cells. We showed that the unwanted cross-regulations displayed a threshold-like effect, and the threshold position varied along with the context of promoters and insulators. We tested the function of insulators in both cascade and sensory switch circuits assembled in episomal plasmid vectors, and showed that the insulation effect was mainly revealed on the first regulatory layer of the cascade circuit. A deviation on the response curve of the sensory switch circuit with or without insulators was observed, but response intensity of some sensory switch circuits were not affected. Therefore, our results provided a general guide on the selection of insulators with varying promoters in episomal synthetic gene circuits in mammalian cells, which may be useful to reduce the effect of the unwanted cross-regulations.

Oral fungal immunomodulatory protein-Flammulina velutipes has influence on pulmonary inflammatory process and potential treatment for allergic airway disease: A mouse model.

BACKGROUND/PURPOSE: House dust mite (HDM) is well known as one of the major indoor allergens that trigger allergic inflammation, especially asthma, and accounts for 85% of all cases. So far, asthma has been thought of as a condition of imbalance between T helper (Th)1 and Th2. Fungal immunomodulatory protein-Flammulina velutipes (FIP-fve) has been seemingly demonstrated to modulate the response to Th1 cytokine production. The aim of this study was to investigate if the oral administration of FIP-fve can inhibit HDM-induced asthma inflammation in the mouse model. METHODS: We divided the mice (female BALB/c, 4-6 weeks) into four groups: the prevention group, which consisted of mice sensitized by HDM (intraperitoneally on Day 1, Day 7, and Day 14, and intranasally on Day 14, Day 17, Day 21, Day 24, and Day 27) fed with FIP-fve from Day 1 to Day 14; the treatment group, which comprised mice that received treatment from Day 14 to Day 28; the positive control (PC, sensitized by HDM fed without FIP-fve) group; and the negative control group (NC, nonsensitized). Airway hyperresponsiveness induced by methacholine challenge was determined using whole-body barometric plethysmography. In addition, cytokines were analyzed from bronchoalveolar lavage fluid and serum. Histopathological studies and Liu's staining method in mice lungs were also performed. RESULTS: The results showed that both pre- and posttreated FIP-fve groups had significantly reduced airway hyperresponsiveness compared with the PC group after methacholine challenge. In addition, a significantly decreased level of HDM-specific immunoglobulin E in serum and decreased production of Th2 cytokines in bronchoalveolar lavage fluid and serum were observed in these two FIP-fve fed groups. Moreover, more decreased amounts of infiltrating inflammatory cells were present in the lungs of FIP-fve fed groups than those of the PC group. CONCLUSION: Oral FIP-fve had an anti-inflammatory effect on the acute phase of the airway inflammatory process induced by HDM in the mouse model and might have a potentially therapeutic role for allergic airway diseases.