Fluorinated Silane-Modified Filtroporation Devices Enable Gene Knockout in Human Hematopoietic Stem and Progenitor Cells.

Intracellular delivery technologies that are cost-effective, non-cytotoxic, efficient, and cargo-agnostic are needed to enable the manufacturing of cell-based therapies as well as gene manipulation for research applications. Current technologies capable of delivering large cargoes, such as plasmids and CRISPR-Cas9 ribonucleoproteins (RNPs), are plagued with high costs and/or cytotoxicity and often require substantial specialized equipment and reagents, which may not be available in resource-limited settings. Here, we report an intracellular delivery technology that can be assembled from materials available in most research laboratories, thus democratizing access to intracellular delivery for researchers and clinicians in low-resource areas of the world.

Recruitment of Plasma Membrane GABA-A Receptors by Submembranous Gephyrin/Collybistin Clusters.

It has been shown that subunit composition is the main determinant of the synaptic or extrasynaptic localization of GABA receptors (GABARs). Synaptic and extrasynaptic GABARs are involved in phasic and tonic inhibition, respectively. It has been proposed that synaptic GABARs bind to the postsynaptic gephyrin/collybistin (Geph/CB) lattice, but not the typically extrasynaptic GABARs.

Collybistin SH3-protein isoforms are expressed in the rat brain promoting gephyrin and GABA-A receptor clustering at GABAergic synapses.

Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3-).

Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells.

Expression of protocadherin-γC4 protein in the rat brain.

It has been proposed that the combinatorial expression of γ-protocadherins (Pcdh-γs) and other clustered protocadherins (Pcdhs) provides a code of molecular identity and individuality to neurons, which plays a major role in the establishment of specific synaptic connectivity and formation of neuronal circuits. Particular attention has been directed to the Pcdh-γ family, for which experimental evidence derived from Pcdh-γ-deficient mice shows that they are involved in dendrite self-avoidance, synapse development, dendritic arborization, spine maturation, and prevention of apoptosis of some neurons. Moreover, a triple-mutant mouse deficient in the three C-type members of the Pcdh-γ family (Pcdh-γC3, Pcdh-γC4, and Pcdh-γC5) shows a phenotype similar to the mouse deficient in whole Pcdh-γ family, indicating that the latter is largely due to the absence of C-type Pcdh-γs.

Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.

The recruitment of inhibitory GABA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42.

In vivo transgenic expression of collybistin in neurons of the rat cerebral cortex.

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ-aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABA receptors (GABA Rs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain.

In vivo clonal overexpression of neuroligin 3 and neuroligin 2 in neurons of the rat cerebral cortex: Differential effects on GABAergic synapses and neuronal migration.

We studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vesicular gamma-aminobutyric acid (GABA) transporter (vGAT) and glutamic acid decarboxylase (GAD)65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent.

Ring finger protein 34 (RNF34) interacts with and promotes γ-aminobutyric acid type-A receptor degradation via ubiquitination of the γ2 subunit.

We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2.

Molecular and functional interaction between protocadherin-γC5 and GABAA receptors.

We have found that the γ2 subunit of the GABA(A) receptor (γ2-GABA(A)R) specifically interacts with protocadherin-γC5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABA(A)R and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 coimmunoprecipitates with GABA(A)Rs.

Differential regulation of the postsynaptic clustering of γ-aminobutyric acid type A (GABAA) receptors by collybistin isoforms.

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses.

Molecular cloning of myostatin gene and characterization of tissue-specific and developmental stage-specific expression of the gene in orange spotted grouper, Epinephelus coioides.

In this article we report the molecular cloning and characterization of a nonmammalian myostatin (growth and differentiation factor-8, MSTN) homolog from the orange spotted grouper (Epinephelus coioides) by polymerase chain reaction (PCR) cloning. The grouper MSTN gene consists of two introns [Intron I (363 bp) and Intron II (811 bp)] flanked by three exons [Exon I (379 bp), Exon II (371 bp) and Exon III (381 bp)]. A full-length cDNA clone (2608 bp) of the MSTN gene (GenBank DQ493889, nucleotide sequence in the coding region identical to GeneBank AY856860) was also isolated.

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence.

Molecular cloning and characterisation of a thioester-containing alpha2-macroglobulin (alpha2-M) from the haemocytes of mud crab Scylla serrata.

Molecular approaches were used to clone thioester-containing alpha2-macroglobulin (alpha2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of alpha2-M was determined by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA clone consists of 5491bp with an open reading frame (ORF) of 4986bp encoding a protein of 1662 amino acids with 22 residues signal sequence.

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids.

Cloning and characterisation of a prophenoloxidase from the haemocytes of mud crab Scylla serrata.

A prophenoloxidase (proPO) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full length of 2663bp, with an open reading frame of 2019bp, a 124-bp 5'-untranslated region, and a 520-bp 3'-untranslated region containing a poly A signal.

Cloning and characterisation of mitochondrial manganese superoxide dismutase (mtMnSOD) from the giant freshwater prawn Macrobrachium rosenbergii.

A cDNA encoding a mitochondrial manganese superoxide dismutase (mtMnSOD) was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) PCR method. Analysis of nucleotide sequence revealed that the mtMnSOD full-length cDNA consists of 1202bp containing an open reading frame of 654bp, which encodes a protein consisting of 218 amino acids including a signal peptide of 16 amino acid residues.