RNA sampling time on postmortem avian carcasses in the wild

Genetic sampling, especially high‐quality RNA from wild avian populations, is challenging in wildlife biology due to rapid RNA degradation. Although carcasses could be a potential RNA source, the optimal postmortem sampling time on the avian carcasses under field conditions remains unclear. Here, we carried out a field experiment on the Qinghai‐Tibet Plateau (QTP) and evaluated the relationship between PMI and RNA degradation in three tissue types (muscle, brain, and liver) of the domestic chicken Gallus gallus domesticus carcasses. In the muscle and brain tissues, we found that the RNA Integrity Number (RIN) of samples collected within 60 h postmortem was more than 7.0, suggesting a high RNA extract quality. The following RNA‐seq experiment demonstrated that gene expression profiles of the samples collected within 36 h postmortem were comparable to those of fresh samples (i.e. 0 h), with a low percentage of differentially expressed genes (< 3.0%) observed between samples at 0 and 36 h postmortem. However, in the liver tissue, RNA samples already degraded at 12 h postmortem, showing low RIN values (< 7.0), different gene expression profiles from fresh samples, and a high percentage of differentially expressed genes (15.6%). Therefore, our study suggests that samples from muscle and brain tissues collected within 36 h postmortem are qualified for RNA‐seq analyses. In contrast, only the fresh RNA samples from liver tissue are qualified. Our study provides a practicable and efficient sampling strategy for the transcriptome study on avian populations under extreme environment such as the QTP

Expression of GA733-Fc Fusion Protein as a Vaccine Candidate for Colorectal Cancer in Transgenic Plants

The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins