Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry

Objective

Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.

Methods

The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.

Results

Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABA_A) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.

Conclusions

Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

     

Genetic Diversity and Population Structure Analyses of Wild Relatives and Cultivated Cowpea (Vigna unguiculata (L.) Walp.) from Senegal Using Simple Sequence Repeat Markers

Cowpea (Vigna unguiculata (L.)) is an important crop for food security in Senegal; therefore, understanding the genetic diversity of local germplasm is relevant for crop improvement and genetic maintenance in the era of climate change. For this purpose, 15 microsatellite markers were used to estimate the genetic diversity of Senegalese cowpea germplasm, including 671 accessions grown in eight regions and 66 wild relatives and intermediate forms (weedy). For the cultivated, the main expected heterozygosity (mHe) ranged between 0.317 (Fatick) and 0.439 (South). A narrow genetic variation between accessions from the different regions was observed with genetic similarity ranging from 0.861 to 0.965 and genetic differentiation indices (Fst) between 0.018 and 0.100. The accessions from southern Senegal (Kédougou, Sédhiou, and Kolda regions) are more diverse than the others. However, the accessions from the North (Saint-Louis) are genetically different from other regions. The diversity analysis in wild relatives from Senegal, which had never been performed before, revealed that the wild/weedy forms remain more diverse than the cultivated with genetic diversity values (He) of 0.389 and 0.480, respectively. STRUCTURE software divided the Senegalese germplasm into five subpopulations. Three of them (i, ii, and iii) included only cultivated accessions from several regions, one (v) mainly from Saint-Louis, and one (iv) the wild/weedy with some cultivated accessions. Our results support the hypothesis that Vigna unguiculata var. spontanea is the wild progenitor of cowpea. The accessions from the South, the northern recession accessions, and the wild/weedy could serve as sources of new genes for the genetic improvement of cowpea in Senegal.

     

Supplemented nutrition decreases helminth burden and increases drug efficacy in a natural host–helminth system

Gastrointestinal (GI) helminths are common parasites of humans, wildlife, and livestock, causing chronic infections. In humans and wildlife, poor nutrition or limited resources can compromise an individual''s immune response, predisposing them to higher helminth burdens. This relationship has been tested in laboratory models by investigating infection outcomes following reductions of specific nutrients. However, much less is known about how diet supplementation can impact susceptibility to infection, acquisition of immunity, and drug efficacy in natural host–helminth systems. We experimentally supplemented the diet of wood mice (Apodemus sylvaticus) with high-quality nutrition and measured resistance to the common GI nematode Heligmosomoides polygyrus. To test whether diet can enhance immunity to reinfection, we also administered anthelmintic treatment in both natural and captive populations. Supplemented wood mice were more resistant to H. polygyrus infection, cleared worms more efficiently after treatment, avoided a post-treatment infection rebound, produced stronger general and parasite-specific antibody responses, and maintained better body condition. In addition, when applied in conjunction with anthelmintic treatment, supplemented nutrition significantly reduced H. polygyrus transmission potential. These results show the rapid and extensive benefits of a well-balanced diet and have important implications for both disease control and wildlife health under changing environmental conditions.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.