Cell cloning-on-the-spot by using an attachable silicone cylinder.

Cell cloning is a laboratory routine to isolate and keep particular properties of cultured cells. Transfected or other genetically modified cells can be selected by the traditional microbiological cloning. In addition, common laboratory cell lines are prone to genotypic drift during their continual culture, so that supplementary cloning steps are often required to maintain correct lineage phenotypes. Here, we designed a silicone-made attachable cloning cylinder, which facilitated an easy and bona fide cloning of interested cells. This silicone cylinder was easy to make, showed competent stickiness to laboratory plastics including culture dishes, and hence enabled secure isolation and culture for days of selected single cells, especially, on the spots of preceding cell-plating dishes under microscopic examination of visible cellular phenotypes. We tested the silicone cylinder in the monoclonal subcloning from a heterogeneous population of a breast cancer cell line, MDA-MB-231, and readily established independent MDA-MB-231 subclones showing different sublineage phenotypes.

Peripheral Neuropathy and Decreased Locomotion of a RAB40B Mutation in Human and Model Animals.

Rab40 proteins are an atypical subgroup of Rab GTPases containing a unique suppressor of the cytokine signaling (SOCS) domain that is recruited to assemble the CRL5 E3 ligase complex for proteolytic regulation in various biological processes. A nonsense mutation deleting the C-terminal SOCS box in the RAB40B gene was identified in a family with axonal peripheral neuropathy (Charcot-Marie-Tooth disease type 2), and pathogenicity of the mutation was assessed in model organisms of zebrafish and Drosophila. Compared to control fish, zebrafish larvae transformed by the human mutant hRAB40B-Y83X showed a defective swimming pattern of stalling with restricted localization and slower motility. We were consistently able to observe reduced labeling of synaptic markers along neuromuscular junctions of the transformed larvae. In addition to the neurodevelopmental phenotypes, compared to normal hRAB40B expression, we further examined ectopic expression of hRAB40B-Y83X in Drosophila to show a progressive decline of locomotion ability. Decreased ability of locomotion by ubiquitous expression of the human mutation was reproduced not with GAL4 drivers for neuron-specific expression but only when a pan-glial GAL4 driver was applied. Using the ectopic expression model of Drosophila, we identified a genetic interaction in which Cul5 down regulation exacerbated the defective motor performance, showing a consistent loss of SOCS box of the pathogenic RAB40B. Taken together, we could assess the possible gain-of-function of the human RAB40B mutation by comparing behavioral phenotypes in animal models; our results suggest that the mutant phenotypes may be associated with CRL5-mediated proteolytic regulation.

Genetic and Clinical Studies of Peripheral Neuropathies with Three Small Heat Shock Protein Gene Variants in Korea.

Small heat shock proteins (sHSPs) are ATP-independent chaperones that help correct the folding of denatured proteins and protect cells from stress. Mutations in HSPB1, HSPB8, and HSPB3 are implicated in inherited peripheral neuropathies (IPNs), such as Charcot-Marie-Tooth disease type 2 (CMT2) and distal hereditary motor neuropathies (dHMN). This study, using whole exome sequencing or targeted gene sequencing, identified 9 pathogenic or likely pathogenic variants in these three sHSP genes from 11 Korean IPN families. Most variants were located in the evolutionally well conserved α-crystallin domain, except for p.P182S and p.S187L in HSPB1. As an atypical case, a patient with dHMN2 showed two compound heterozygous variants of p.R127Q and p.Y142H in HSPB1, suggesting a putative case of recessive inheritance, which requires additional research to confirm. Three HSPB8 variants were located in the p.K141 residue, which seemed to be a mutational hot spot. There were no significant differences between patient groups, which divided by sHSP genes for clinical symptoms such as onset age, severity, and nerve conduction. Early-onset patients showed a tendency of slightly decreased sensory nerve conduction values compared with late-onset patients. As a first Korean IPN cohort study examining sHSP genes, these results will, we believe, be helpful for molecular diagnosis and care of patients with CMT2 and dHMN.