Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides ^{1 ,2-5}. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.     

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads.However,transformation of environmental strains is much more difficult than that of domesticated strains.Here,we report the development of an efficient and robust electroporation-based transformation system for marine-derived Bacillus marinus B-9987,which is a macrolactin antibiotics producer and a very promising biological control agent against fungal plant diseases.The transformation efficiency was greatly enhanced 103-fold by using unmethylated plasmid to bypass modification-restriction barrier,and using glycine betaine to protect cells from electrical damages during electroporation.Addition of HEPES and 2 mmol L?1MgCl2 further improved the efficiency by additional 2-fold,with a maximum value of 7.1×104 cfu/μg pHT3101.To demonstrate the feasibility and efficiency of the protocol,a green fluorescent protein reporter system was constructed;furthermore,phosphopantetheinyl transferase gene sfp,which is essential to the biosynthesis of polyketides and nonribosomal peptides,was overexpressed in B-9987,leading to increased production of macrolactin A by about 1.6-fold.In addition,this protocol is also applicable to marine-derived Bacillus licheniforms EI-34-6,indicating it could be a reference for other undomesticated Bacillus strains.To our knowledge,this is the first report regarding the transformation of marine-derived Bacillus strain.     

A transportable,inexpensive electroporator for in utero electroporation

Electroporation is a useful technique to study gene function during development but its broad application is hampered due to the expensive equipment needed. We describe the construction of a transportable, simple and inexpensive electroporator delivering square pulses with varying length and amplitude. The device was successfully used for in utero electroporation in mouse with a performance comparable to that of commercial products.     

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system ^1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex ^6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method ⁹. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice ^10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA ¹². These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.     

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.     

Periostin,a neurite outgrowth‐promoting factor,is expressed at high levels in the primate cerebral cortex

Periostin (POSTN or osteoblast specific factor) is an extracellular matrix protein originally identified as a protein highly expressed in osteoblasts. Recently, periostin has been reported to function in axon regeneration and neuroprotection. In the present study, we focused on periostin function in cortical evolution. We performed a comparative gene expression analysis of periostin between rodents (mice) and primates (marmosets and macaques). Periostin was expressed at higher levels in the primate cerebral cortex compared to the mouse cerebral cortex. Furthermore, we performed overexpression experiments of periostin in vivo and in vitro. Periostin exhibited neurite outgrowth activity in cortical neurons. These results suggested the possibility that prolonged and increased periostin expression in the primate cerebral cortex enhances the cortical plasticity of the mammalian cerebral cortex.     

Pulsed electric field (PEF) as an intensification pretreatment for greener solvent lipid extraction from microalgae

Microalgae, with their high lipid content, are a promising feedstock for renewable fuels. Traditionally, human and environmentally toxic solvents have been used to extract these lipids, diminishing the sustainability of this process. Herein, pulsed electric field technology was utilized as a process intensification strategy to enhance lipid extraction from Ankistrodesmus falcatus wet biomass using the green solvent, ethyl acetate. The extraction efficiency for ethyl acetate without PEF was lower (83–88%) than chloroform. In addition, the ethyl acetate exhibited a 2‐h induction period, while the chloroform showed no time dependence. Utilizing PEF technology resulted in 90% of the cells being lysed and a significant enhancement in the rate of lipid recovery using ethyl acetate. The increase in lipid recovery was due to the presence of the electric field and not due to temperature effects. The PEF technology uses less energy than other PEF systems reported in the literature. Biotechnol. Bioeng. 2013; 110: 1605–1615. © 2013 Wiley Periodicals, Inc.