Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.New modifications to the accelerator chamber have enhanced the gene gun''s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene''s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.     

Improved Biolistic Transfection of Hair Cells

Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca²⁺-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.     

新型"脂质-HAP-DNA复合体"的制备和用于真核细胞基因转染研究

为寻找一种简单、经济、有效的DNA递送系统用于基因转染和基因治疗,制备了表面电荷为正电的纳米HAP,与表面电荷为负电的DNA结合形成DNA-HAP复合物,采用逆向蒸发法,用卵磷脂、DOPE和胆固醇制备成脂质体包封DNA-HAP复合物形成脂质-HAP-DNA复合体,脂质体和HAP对照,对所形成的脂质-HAP-DNA复合体(LHD)的特性、包封率、转染Hela细胞的效果进行初步检测研究。所获得的脂质-HAP-DNA复合体呈球形、平均粒径为643nm;平均包封率达11.67%,为中性脂质体;能有效转染真核细胞。该方法可作为提高基因转染效果的简单、经济、有效的手段之一,也为进一步提高非病毒载体的转染效率提供了一个思路。     

量子点在基因转染中的应用研究进展

量子点因其独特的纳米尺寸效应、光学特性和生物相容性,既能作为纳米载体与目的基因结合,又能作为纳米荧光标记物跟踪记录其在转染过程中的位置,给基因工程的发展带来了新的契机。在阐述量子点用于基因转染的优势、标记基因的方法等基础上,作者系统综述了量子点在基因转染中的应用,并对其发展趋势和应用前景进行了展望。     

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5’ mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work¹¹, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.     

CHO-K1细胞中基因瞬时转染的条件优化

目的:以CHO-K1细胞为宿主基因瞬时转染条件的优化.方法:以GFP(Green Fluorescence Protein)为报告基因,考察了DNA∶PEI比例、DNA用量及血清的加入对CHO-K1细胞的转染效率和细胞数目的影响.结果:DNA∶PEI=1∶2(w/w)、2 gDNA/106 cells时,转染结果最优;血清的加入可降低细胞转染效率.结论:在CHO-K1细胞中进行瞬时转染的最佳条件为DNA∶PEI=1∶2(w/w)、2 gDNA/106 cells,及血清的加入抑制细胞转染.     

基因枪法转基因水稻中hpt基因稳定遗传

基因枪转化将潮霉素磷酸转移酶基因（ｈｐｔ）导入粳稻品种７７１７０,获得可育的转基因植株,研究外源基因遗传的稳定性。自交后代（Ｔ１和Ｔ２）经潮霉素筛选获得抗性植株和敏感植株,分子鉴定结果表明抗性植株带有ｈｐｔ基因,而敏感植株中没有ｈｐｔ基因存在。Ｔ１和Ｔ２代中潮霉素抗性表现为显性单基因位点的遗传方式,符合孟德尔分离规律,并得到分子鉴定结果的证实。Ｓｏｕｔｈｅｒｎ杂交结果显示,ｈｐｔ基因多拷贝整合在水     

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals ^1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality ^3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area ^5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish ⁷, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.     

Rotenone Induces Cell Death of Cholinergic Neurons in an Organotypic Co-Culture Brain Slice Model

In Alzheimer and Parkinson’s disease cell death of cholinergic and dopaminergic neurons are characteristic hallmarks, respectively. It is well established that rotenone, an inhibitor of complex I of the electron transport chain, induces cell death of dopaminergic neurons, however, not much is known on the effects of rotenone on cholinergic neurons. The aim of the present study was to evaluate the effects of rotenone on cholinergic neurons in an organotypic in vitro brain co-slice model. When co-cultures were treated with 10 μM rotenone for 24 h a significantly decreased number of cholinergic neurons was found in the basal nucleus of Meynert but not in the dorsal striatum. This cell death exhibited apoptotic DAPI-positive malformed nuclei and enhanced TUNEL-positive cells. In summary, inhibition of complex I of the electron transport chain may play a role in neurodegeneration of cholinergic neurons.     

睾丸内注射pEGFP-N1体内转染精子并在山羊早期胚胎成功表达

目的探讨睾丸内注射法pEGFP-N1在精细胞的整合和在早期胚胎中表达.方法选择4头本地山羊,双侧睾丸注射不同剂量质粒DNA pEGFP-N1,注射后PCR和Southern杂交检测pEGEP-N1在精子中的整合.结果pEGFP-N1整合到精子基因组中,转染效率最高发生在注射后第40天,转染阳性率最高为81%;绿色荧光蛋白在精子及其体外受精的部分胚胎中表达,胚胎阳性率最高的达66.7%.结论通过睾丸内注射pEGFP-N1能整合进入精子基因组,并能通过体外受精在山羊早期胚胎中表达;睾丸内注射法可能是一种可行、简单并利于推广的制备转基因山羊的方法.     

Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents

This study compared six commercially available reagents (Arrest-In, ExpressFect, FuGENE HD, jetPEI, Lipofectamine 2000, and SuperFect) for gene transfection. We examined the efficiency and cytotoxicity using nine different cell lines (MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma), and primary cells (HEKn human epidermal keratinocytes) with two different plasmid DNAs encoding luciferase or β-galactosidase in the presence or absence of serum. Maximal transfection efficiency in MC3T3-E1, C3H10T1/2, HeLa, C2C12, Hep G2, and HCT116 was seen using FuGENE HD, in PT-30, 4T1, and HEKn was seen using Arrest-In, and in MCF-7 was seen using jetPEI. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetPEI and ExpressFect. These results suggest that FuGENE HD is the most preferred transfection reagent for many cell lines, followed by Arrest-In and jetPEI. These results may be useful for improving nonviral gene and cell therapy applications.     

不同修饰对壳聚糖转基因效果的影响

目的:探讨不同修饰的壳聚糖包裹的质粒(Chi-DNA)纳米复合物在口服发送中外源 基因在消化道中的表达差异.方法:分别使用明胶、海藻酸钠、PEG及乙酰化修饰包裹含Lac Z的质粒pCMVa的壳聚糖纳米颗粒,通过口服发送后经X-gal染色检测目的基因在小鼠体内的 表达.结果:修饰后的Chi-DNA纳米复合物都能抵抗胃酸的降解0.5h以上 ,其中PEG及乙酰化 修饰的Chi-DNA纳米复合物在胃酸处理1h时仍有部分残留.X-gal染色显示,修饰后的Chi-DN A纳米复合物都有a半乳糖苷酶的表达,其中PEG、海藻酸钠修饰的Chi-DNA纳米复合物在鼠 胃和小肠中表达量最高.结论:PEG、海藻酸钠修饰的Chi-DNA纳米复合物有望成为高效的基因治疗用非病毒口服发送系统.     

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson''s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.     

Lipopolyamine-Mediated Transfection Allows Gene Expression Studies in Primary Neuronal Cells

A simple and efficient transfection technique based on lipopolyamine-coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans-acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.     

Effect of CDP-Choline on Hypocapnic Neurons in Culture

Neuronal cultures from chick embryo cerebral hemispheres were protected against a hypocapnic injury by adding to their growth medium 10(-6)M CDP-choline before or after the injury. The protection obtained with CDP-choline was analyzed by a morphometric analysis and showed that pretreatment of neuronal cultures with CDP-choline maintained the number of cell aggregates and of primary neuronal processes at control values after hypocapnic shock. Various experiments showed that the intact molecule was responsible for the protective action, since pretreatment with different concentrations of various nucleosides and nucleotides (up to 10(-5) M), choline, and phosphorylcholine was without protective effect. The addition of CDP-choline after the hypocapnic injury resulted in a protection of the cultures as shown by morphological observation. Incubation of neurons with radioactive choline showed that hypocapnia increased the incorporation of the label into phospholipids whereas the presence of CDP-choline reduced it. The de novo synthesis of choline was affected by neither hypocapnia nor CDP-choline treatment. The results indicate that CDP-choline may have the capacity to protect neurons under conditions of basic pH and that cellular proliferation may be stimulated by the compound.     

提高精子转染外源DNA效率的山羊精液冷冻方法

目的筛选一种既提高精子转染外源DNA效率,又保持解冻后精子活力的山羊精液冷冻—解冻方法。方法应用正交设计L9(34),因素分别为稀释液种类、稀释比例、降温时间和解冻液,每个因素选择3个水平,检测和比较解冻后精子转染外源DNA效率和精子活力。结果所选冷冻—解冻各因素对精液转染效率影响不显著[F(8,18)=1.032,P=0.449];平衡时间对冷冻—解冻活力影响极显著[F(2,24)=9.972,P=0.001],平衡1h极显著小于平衡2 h和4 h的精液活力(P=0.003,P=0.000),以平衡4 h最好。用筛选的冷冻—解冻方法处理精液,解冻后精子的活力极明显降低(P=0.002);生存指数降低,GOT释放量增加,菌落数减少,与鲜精相比差异显著(P=0.018;P=0.016;P=0.018);精子畸形率增加,顶体完整率降低,与鲜精相比差异不显著(P=0.494;P=0.084)。结论优化了提高精子转染外源DNA效率的山羊精液冷冻—解冻方法。     

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel''s cartilage, nasal glands, tongue, and ear.     

Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10⁻⁷, 10⁻⁶ and 10⁻⁵ M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.