Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice

Our previous studies have demonstrated that Fam20 C promotes differentiation and mineralization of odontoblasts,ameloblasts,osteoblasts and osteocytes during tooth and bone development.Ablation of the Fam20 C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice.However,control and regulation of the expression of Fam20 C are still unknown.In this study,we generated a transgenic reporter model which expresses green fluorescence protein（GFP） driven by the Fam20 C promoter.Recombineering was used to insert a 16 kb fragment of the mouse Fam20 C gene（containing the 15 kb promoter and 1.1 kb of exon 1） intoa pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence.GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice.Fluorescence was evident in the bone and teeth as early as E17.5.The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth.The expression of GFP was significantly reduced in teeth,alveolar bone and muscle by 8 weeks of age.We also observed colocalization of the GFP signal with the Fam20 C antibody in postnatal 1- and 7-day-old animals.Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20 C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-Glc NAcylation in HEK293 T cells.The fluorescent reporter mainly consists of a reporter(green fluorescent protein(GFP)),an internal reference(red fluorescent protein),and an operator(neuronal differentiation 1),which serves as a"sweet switch"to control GFP expression in response to cellular OGlc NAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular OGlc NAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-Glc NAcylation in He La and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for conducting gene transcription studies.

Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize

Genome editing by clustered regularly interspaced short palindromic sequences(CRISPR)/CRISPRassociated protein 9(Cas9)has revolutionized functional gene analysis and genetic improvement.While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation,this approach has not been well established in seed crops.Here,we established the seed fluorescence reporter(SFR)-assisted CRISPR/Cas9 systems in maize(Zea mays L.),using the red fluorescent Ds RED protein expressed in the endosperm(En-SFR/Cas9),embryos(Em-SFR/Cas9),or both tissues(Em/En-SFR/Cas9).All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out.We describe several case studies of the implementation of En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9 to identify plants not harboring the genomeediting cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries,and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9.SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

The Fold-Illuminator:A low-cost,portable,and disposable incubator-illuminator device

Fluorescent reporters have revolutionized modern applications in the fields of molecular and synthetic biology,enabling applications ranging from education to point-of-care diagnostics.Past advancements in these fields have primarily focused on improving reaction conditions,the development of new applications,and the broad dissemination of these technologies.However,field and classroom-based applications have remained limited in part due to the nature of fluorescent signal detection,which often requires the use of costly lab equipment to observe and quantify fluorescence readouts.Users without access to laboratory equipment rely on qualitative assessments of fluorescence,a process that remains highly variable from user-to-user even within the same classroom.To overcome this challenge,we have developed a foldable illuminator and incubator device to support field-applications of synthetic biology-based biosensors for education and diagnostics.The Fold-Illuminator is an affordable,portable,and recyclable device that allows for the visible detection of fluorescent biomolecules.The Fold-Illuminator’s design allows for assembly in under 10 min,a user can then utilize the optional heating element to incubate biochemical reactions and visualize fluorescence outputs in a defined and light-controlled environment.Interchangeable LED strips and light-filtering screens provide modularity to pair with the fluorescence wavelengths of interest.The user can then unfold the device for convenient storage,transport,or even recycling.The cost for the Fold-Illuminator is$5.58 USD and is compatible with an optional heating element for an additional$3.98 cost,with potential for further reductions in cost for larger quantities.Open-source templates for cutting device parts from paper stock are provided for both printing and cutting by hand;cutting can also be achieved with consumer-grade smart cutting machines such as the Cricut®.Combined with the broad applications of fluorescent reporters,the Fold-Illuminator has the potential to improve access to fluorescence visualization and quantification for new users as well as emerging field applications.