Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.     

Evaluation of DAPI as a Fluorescent Probe for DNA in Viable Petunia Protoplasts

4',6-Diamidino-2-phenyl-indole (DAPI), is a fluorescent probe that specifically and quantitatively stains DNA. Electroporation of viable Petunia protoplasts in the presence of DAPI revealed integral fluorescence that was similar for both the electroporated and fixed protoplasts. indicating quantitative staining of DNA. DAPI fluorescence was localized in the nuclei of viable protoplasts of Petunia. Protoplasts had a short term viability of 56-65% of the control (non-electroporated. unstained) protoplasts as determined by fluorescein diacetate staining 24 hr following electroporation in the presence of DAPI. The majority (84% of the number originally cultured) of these protoplasts subjected to electroporation were able to form a cell wall, but most did not form microcalli because they were blocked in cell division. The three week plating efficiency for protoplasts exposed to DAPI was 4% of the original number of protoplasts initially cultured compared to 30% for the control. DAPI should not be used as a fluorescent probe for plant protoplasts when the protoplasts are cultured for sustained growth because the levels of DAPI required to obtain quantitative staining of the DNA resulted in inhibition of the cell cycle. DAPI may, however, be used as a fluorescent DNA probe for short term (24 hr) studies.     

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.     

以Zeocin抗性基因为选择标记的Candida glycerinogenes遗传转化

为从分子水平上阐释产甘油假丝酵母(Candida glycerinogenes)高产甘油机理,建立一种方便可行的遗传转化系统是十分必要的。与G418和潮霉素等抗生素相比,Zeocin抗生素对C.glycerinogenes具有较低的致死浓度。以pGAPZb作为构建整合载体的骨架,以Zeocin抗性基因作为选择标记,以URA3基因作为整合位点,构建了C.glycerinogenes整合载体pGA-CU。整合载体经过限制酶线性化后用作转化载体,基于电击转化的方法成功获得了抗Zeocin的转化子并经过PCR分析进一步确证。通过优化电击转化的参数,获得了较为稳定的转化效率,基于这一技术的转化效率每微克DNA可获得120个转化子。为进一步研究该菌株的遗传背景和代谢机理奠定了基础。     

N‐cadherin/catenin–based costameres in cultured chicken cardiomyocytes

N‐cadherin is a member of the Ca²⁺‐dependent cell adhesion molecules and plays an important role in the assembly of the adherens junction in chicken cardiomyocytes. In addition to being present at the cell‐cell junction, N‐cadherin is associated with costameres in extrajunctional regions. The significance of the N‐cadherin‐associated costameres and whether catenins are components of costameres in chicken cardiomyocytes are not known. In this study, double‐labeling immunofluorescence microscopy was used to determine the extrajunctional distribution of both N‐cadherin and its cytoplasmic associated proteins, α‐ and β‐catenins, and their relationship to myofibrillar Z‐disc α‐actinin. N‐cadherin, α‐, and β‐catenins were all found to be present at the extrajunctional region and, in some cases, were codistributed with myofibrillar α‐actinin exhibiting a periodic staining pattern. Confocal microscopy confirmed that both N‐cadherin and β‐catenin colocalized with peripheral myofibrillar α‐actinin on the dorsal surface of cardiomyocytes as components of the costameres. Intracellular application of antibodies specific for the cytoplasmic portions of N‐cadherin, α‐, and β‐catenin, either by electroporation or microinjection, resulted in myofibril disorganization and disassembly. These results suggest the existence of N‐cadherin/catenin‐based costameres in the dorsal surface of cultured chicken cardiomyocytes in addition to the integrin/vinculin‐based costameres found in the ventral surface and indicate that the former set of costameres is essential for cardiac myofibrillogenesis. J. Cell. Biochem. 75:93–104, 1999. © 1999 Wiley‐Liss, Inc.     

棒状类细菌电击转化中多种条件对转化效率的影响

以质粒PXZl0145电击转化不同棒状类细菌菌株,研究了影响电击转化效率的诸个因素,在含4%甘氨酸的培养基中生长至对数前期的菌体最适用于电击转化,当以同源DNA进行电击转化时,1．μgDNA中转化效率最高可达到8×1O⁶转化子,但用异源DNA时,转化效率要比前者低10²～10³倍。     

植物体细胞原生质体遗传转化研究

重点介绍了植物体细胞原生质体遗传转化的方法和当前已经取得的成果,同时提出了目前原生质体遗传转化中存在的问题,展望了今后的工作重点。植物原生质体遗传转化的方法主要有：PEG介导转化法、电击穿孔转化法、脂质体介导转化法、农杆菌共培养转化法等。