An undergraduate laboratory experience using CRISPR-cas9 technology to deactivate green fluorescent protein expression in Escherichia coli.

Undergraduates learn that gene editing in diverse organisms is now possible. How targeted manipulation of genes and genomes is utilized in basic science and biomedicine to address biological questions is challenging for undergraduates to conceptualize. Thus, we developed a lab experience that would allow students to be actively engaged in the full process of design, implementation of a gene editing strategy, and interpretation of results within an 8-week lab period of a Genetics course. The laboratory experience combines two transformative biotechnology tools; the utilization of green fluorescent protein (GFP) as a diagnostic marker of gene expression and the fundamentals and specificity of Clustered Regularly Interspaced Short Palindromic Repeats-cas9 (CRISPR-cas9) gene editing in bacterial cells. The students designed and constructed plasmids that express single guide RNA targeted to GFP, expressed the sgRNA and cas9 in bacteria cells, and successfully deactivated GFP gene expression in the bacterial cells with their designed CRISPR-cas9 tools. Student assessment revealed most students achieved student learning objectives. We conclude this lab experience is an effective and accessible method for engaging students in the scientific practices, knowledge and challenges revolving targeted CRISPR-cas9 gene manipulation. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 145-155, 2019.

Amphiphilic siRNA Conjugates for Co-Delivery of Nucleic Acids and Hydrophobic Drugs.

Combination therapy of nucleic acids and chemical drugs for cancer treatment is a promising strategy to enhance the therapeutic efficacy by simultaneously regulating multiple troublesome pathways. In this study, we report on polyethylene glycol-siRNA-polycaprolactone (PEG-siRNA-PCL) micelles that encapsulate hydrophobic drugs for efficient co-delivery of siRNA and drugs to cancer cells. Amphiphilic PEG-siRNA-PCL copolymers were synthesized by annealing antisense siRNA-PCL conjugates with sense siRNA-PEG conjugates. After paclitaxel encapsulation, PEG-siRNA-PCL micelles containing antiapoptotic Bcl-2-specific siRNA were stabilized with linear polyethylenimine via electrostatic interactions. Stabilized PEG-siRNA-PCL micelles showed superior anticancer effects, assessed by caspase-3 activity analysis, apoptotic cell staining, and a cytotoxicity test, to those of paclitaxel-free PEG-siRNA-PCL micelles and unmodified siRNAs. The strong anticancer activity of paclitaxel-incorporated siRNA micelles can be attributed to the synergistic effect of Bcl-2 siRNA and paclitaxel. This work provides an efficient co-delivery platform for combination anticancer therapy with siRNA and chemotherapy.

Adult Brain Serotonin Deficiency Causes Hyperactivity, Circadian Disruption, and Elimination of Siestas.

UNLABELLED: Serotonin (5-HT) is a crucial neuromodulator linked to many psychiatric disorders. However, after more than 60 years of study, its role in behavior remains poorly understood, in part because of a lack of methods to target 5-HT synthesis specifically in the adult brain. Here, we have developed a genetic approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system by stereotaxic injection of an adeno-associated virus expressing Cre recombinase (AAV-Cre) into the midbrain/pons of mice carrying a loxP-conditional tryptophan hydroxylase 2 (Tph2) allele. We investigated the behavioral effects of deficient brain 5-HT synthesis and discovered a unique composite phenotype. Surprisingly, adult 5-HT deficiency did not affect anxiety-like behavior, but resulted in a robust hyperactivity phenotype in novel and home cage environments. Moreover, loss of 5-HT led to an altered pattern of circadian behavior characterized by an advance in the onset and a delay in the offset of daily activity, thus revealing a requirement for adult 5-HT in the control of daily activity patterns. Notably, after normalizing for hyperactivity, we found that the normal prolonged break in nocturnal activity (siesta), a period of rapid eye movement (REM) and non-REM sleep, was absent in all animals in which 5-HT deficiency was verified. Our findings identify adult 5-HT as a requirement for siestas, implicate adult 5-HT in sleep-wake homeostasis, and highlight the importance of our adult-specific 5-HT-synthesis-targeting approach in understanding 5-HT's role in controlling behavior. SIGNIFICANCE STATEMENT: Serotonin (5-HT) is a crucial neuromodulator, yet its role in behavior remains poorly understood, in part because of a lack of methods to target specifically adult brain 5-HT synthesis. We developed an approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system. Using this technique, we discovered that adult 5-HT deficiency led to a novel compound phenotype consisting of hyperactivity, disrupted circadian behavior patterns, and elimination of siestas, a period of increased sleep during the active phase. These findings highlight the importance of our approach in understanding 5-HT's role in behavior, especially in controlling activity levels, circadian behavior, and sleep-wake homeostasis, behaviors that are disrupted in many psychiatric disorders such as attention deficit hyperactivity disorder.

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

Tissue-Specific Cell Cycle Indicator Reveals Unexpected Findings for Cardiac Myocyte Proliferation.

RATIONALE: Discerning cardiac myocyte cell cycle behavior is challenging owing to commingled cell types with higher proliferative activity. OBJECTIVE: To investigate cardiac myocyte cell cycle activity in development and the early postnatal period. METHODS AND RESULTS: To facilitate studies of cell type-specific proliferation, we have generated tissue-specific cell cycle indicator BAC transgenic mouse lines. Experiments using embryonic fibroblasts from CyclinA2-LacZ-floxed-EGFP, or CyclinA2-EGFP mice, demonstrated that CyclinA2-ßgal and CyclinA2-EGFP were expressed from mid-G1 to mid-M phase. Using Troponin T-Cre;CyclinA2-LacZ-EGFP mice, we examined cardiac myocyte cell cycle activity during embryogenesis and in the early postnatal period. Our data demonstrated that right ventricular cardiac myocytes exhibited reduced cell cycle activity relative to left ventricular cardiac myocytes in the immediate perinatal period. Additionally, in contrast to a recent report, we could find no evidence to support a burst of cardiac myocyte cell cycle activity at postnatal day 15. CONCLUSIONS: Our data highlight advantages of a cardiac myocyte-specific cell cycle reporter for studies of cardiac myocyte cell cycle regulation.

Chitosan Nanoparticles for SiRNA Delivery In Vitro.

RNA interference, the process in which small interfering RNAs (SiRNAs) silence a specific gene and thus inhibit the associated protein, has opened new doors for the treatment of a wide range of diseases. However, efficient delivery of SiRNAs remains a challenge, especially due to their instability in biological environments and their inability to cross cell membranes. To protect and deliver SiRNAs to mammalian cells, a variety of polymeric nanocarriers have been developed. Among them, the polysaccharide chitosan has generated great interests. This derivative of natural chitin is biodegradable and biocompatible, and can complex SiRNAs into nanoparticles on account of its positive charges. However, chitosan presents some limitations that need to be taken into account when designing chitosan/SiRNA nanoparticles. Here, we describe a method to prepare SiRNA/chitosan nanoparticles with high gene silencing efficiency and low cytotoxicity by using the ionic gelation technique.

Somatostatin and its 2A receptor in dorsal root ganglia and dorsal horn of mouse and human: expression, trafficking and possible role in pain.

BACKGROUND: Somatostatin (SST) and some of its receptor subtypes have been implicated in pain signaling at the spinal level. In this study we have investigated the role of SST and its sst2A receptor (sst2A) in dorsal root ganglia (DRGs) and spinal cord. RESULTS: SST and sst2A protein and sst2 transcript were found in both mouse and human DRGs, sst2A-immunoreactive (IR) cell bodies and processes in lamina II in mouse and human spinal dorsal horn, and sst2A-IR nerve terminals in mouse skin. The receptor protein was associated with the cell membrane. Following peripheral nerve injury sst2A-like immunoreactivity (LI) was decreased, and SST-LI increased in DRGs. sst2A-LI accumulated on the proximal and, more strongly, on the distal side of a sciatic nerve ligation. Fluorescence-labeled SST administered to a hind paw was internalized and retrogradely transported, indicating that a SST-sst2A complex may represent a retrograde signal. Internalization of sst2A was seen in DRG neurons after systemic treatment with the sst2 agonist octreotide (Oct), and in dorsal horn and DRG neurons after intrathecal administration. Some DRG neurons co-expressed sst2A and the neuropeptide Y Y1 receptor on the cell membrane, and systemic Oct caused co-internalization, hypothetically a sign of receptor heterodimerization. Oct treatment attenuated the reduction of pain threshold in a neuropathic pain model, in parallel suppressing the activation of p38 MAPK in the DRGs CONCLUSIONS: The findings highlight a significant and complex role of the SST system in pain signaling. The fact that the sst2A system is found also in human DRGs and spinal cord, suggests that sst2A may represent a potential pharmacologic target for treatment of neuropathic pain.

Flow cytometry-based cell type-specific assessment of target regulation by pulmonary siRNA delivery.

Pulmonary siRNA delivery has attracted strong interest and has been reported to successfully mediate target gene knockdown in a number of disease models. However, the nature of the epithelial cells that eventually take up siRNA and the question if other lung cell types may also be transfected have so far been neglected. Therefore, we describe here a flow cytometry-based method using transgenic enhanced green fluorescence protein-expressing mice (EGFP mice) for the differentiation of transfected lung cell populations based on their antigen expression.

MYMIV-AC2, a geminiviral RNAi suppressor protein, has potential to increase the transgene expression.

Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.

siRNA-aptamer chimeras on nanoparticles: preserving targeting functionality for effective gene silencing.

SiRNA-aptamer chimeras are emerging as a highly promising approach for cell-type specific delivery of siRNA due to the outstanding targeting capability of aptamers and the compatibility of chimeras with native ribonuclease (Dicer) processing. For efficient RNA interference (RNAi), however, additional challenges must be addressed, in particular how to get siRNA out of the endosome after cell entry and how to preserve aptamer targeting specificity when chimeras are combined with delivery carriers. Here, we report a rationally designed nanoparticle vector that simultaneously displays large surface area for high siRNA payload, exposed aptamer for specific targeting, proton sponge effect for endosome escape, and fluorescence for imaging and quantification. A key concept of this work is to graft chimeras onto nanoparticle surface via a two-step process: first immobilizing siRNA onto nanoparticle via noncovalent interactions to facilitate intracellular unpackaging and reduce nanoparticle surface charge (avoiding nonspecific electrostatic interactions between aptamers and nanoparticles) and then coupling siRNA and aptamer with retained conformation and high accessibility. Compared with conventional one-step adsorption of siRNA-aptamer chimeras onto nanoparticles with random orientations and conformations, which does not elicit much improved RNAi effect than nontargeted nanoparticle-siRNA complexes (∼6-8% improvement of the total cell population), under the same RNA concentration our approach shows selective gene silencing and enables 34% more silenced cells of the total cell population over nontargeted nanoparticle-siRNA complexes. This remarkable difference in RNAi efficiency using nanoparticle-chimera complexes is directly related to cell uptake discrepancy resulting from aptamer conformation on the nanoparticle surface (intact vs random).

Differential expression of NLRP3 among hematopoietic cells.

Although the importance of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in health and disease is well appreciated, a precise characterization of NLRP3 expression is yet undetermined. To this purpose, we generated a knock-in mouse in which the Nlrp3 coding sequence was substituted for the GFP (enhanced GFP [egfp]) gene. In this way, the expression of eGFP is driven by the endogenous regulatory elements of the Nlrp3 gene. In this study, we show that eGFP expression indeed mirrors that of NLRP3. Interestingly, splenic neutrophils, macrophages, and, in particular, monocytes and conventional dendritic cells showed robust eGFP fluorescence, whereas lymphoid subsets, eosinophils, and plasmacytoid dendritic cells showed negligible eGFP levels. NLRP3 expression was highly inducible in macrophages, both by MyD88- and Trif-dependent pathways. In vivo, when mice were challenged with diverse inflammatory stimuli, differences in both the number of eGFP-expressing cells and fluorescence intensity were observed in the draining lymph node. Thus, NLRP3 levels at the site of adaptive response initiation are controlled by recruitment of NLRP3-expressing cells and by NLRP3 induction.

Persistence of different forms of transient RNAi during apoptosis in mammalian cells.

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2-3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events.

Nonfunctional regulatory T cells and defective control of Th2 cytokine production in natural scurfy mutant mice.

Foxp3(+) regulatory T cells (Tregs) are crucial for preventing autoimmunity. We have demonstrated that depletion of Foxp3(+) Tregs results in the development of a scurfy-like disease, indicating that Foxp3(-) effector T cells are sufficient to induce autoimmunity. It has been postulated that nonfunctional Tregs carrying potentially self-reactive T cell receptors may contribute to scurfy (sf) pathogenesis due to enhanced recognition of self. Those cells, however, could not be identified in sf mutants due to the lack of Foxp3 protein expression. To address this issue, we crossed the natural sf mouse mutant with bacterial artificial chromosome transgenic DEREG (depletion of regulatory T cells) mice. Since DEREG mice express GFP under the control of an additional Foxp3 promoter, those crossings allowed proving the existence of "would-be" Tregs, which are characterized by GFP expression in the absence of functional Foxp3. Sf Tregs lost their in vitro suppressive capacity. This correlated with a substantial reduction of intracellular cAMP levels, whereas surface expression of Treg markers was unaffected. Both GFP(+) and GFP(-) sf cells produced high amounts of Th2-type cytokines, reflected also by enhanced Gata-3 expression, when tested in vitro. Nevertheless, sf Tregs could be induced in vitro, although with lower efficiency than DEREG Tregs. Transfer of GFP(+) sf Tregs, in contrast to GFP(-) sf T cells, into RAG1-deficient animals did not cause the sf phenotype. Taken together, natural and induced Tregs develop in the absence of Foxp3 in sf mice, which lack both suppressive activity and autoreactive potential, but rather display a Th2-biased phenotype.