Multiplexing Bioluminescent and Fluorescent Reporters to Monitor Live Cells

Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein

Paternal chromosome elimination of inducer triggers induction of double haploids in Brassica napus

A synthetic octoploid rapeseed, Y3380, induces maternal doubled haploids when used as a pollen donor to pollinate plant. However, the mechanism underlying doubled haploid formation remains elusive. We speculated that double haploid induction occurs as the inducer line’s chromosomes pass to the maternal egg cell, and the zygote is formed through fertilization. In the process of zygotic mitosis, the paternal chromosome is specifically eliminated. Part of the paternal gene might have infiltrated the maternal genome through homologous exchange during the elimination process. Then, the zygote haploid genome doubles (early haploid doubling, EH phenomenon), and the doubled zygote continues to develop into a complete embryo, finally forming doubled haploid offspring. To test our hypothesis, in the current study, the octoploid Y3380 line was back bred with the 4122-cp4-EPSPS exogenous gene used as a marker into hexaploid Y3380-cp4-EPSPS as paternal material to pollinate three different maternal materials. The fertilization process of crossing between the inducer line and the maternal parent was observed 48 h after pollination, and the fertilization rate reached 97.92% and 98.72%. After 12 d of pollination, the presence of cp4-EPSPS in the embryo was detected by in situ PCR, and at 13–23 d after pollination, the probability of F1 embryos containing cp4-EPSPS gene was up to 97.27%, but then declined gradually to 0% at 23–33 d. At the same time, the expression of cp4-EPSPS was observed by immunofluorescence in the 3rd to 29th day embryo. As the embryos developed, cp4-EPSPS marker genes were constantly lost, accompanied by embryonic death. After 30 d, the presence of cp4-EPSPS was not detected in surviving embryos. Meanwhile, SNP detection of induced offspring confirmed the existence of double haploids, further indicating that the induction process was caused by the loss of specificity of the paternal chromosome. The tetraploid-induced offspring showed infiltration of the induced line gene loci, with heterozygosity and homozygosity. Results indicated that the induced line chromosomes were eliminated during embryonic development, and the maternal haploid chromosomes were synchronously doubled in the embryo. These findings support our hypothesis and lay a theoretical foundation for further localization or cloning of functional genes involved in double haploid induction in rapeseed

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field