Transfection

Abstract: This lab was conducted to determine the efficiency of transfection in HEK293 cells using cell culture and lipofectamine by growing the HEK293 cells out in growth medium, and then passaging them into 24 well plate at 50% confluency. After the passaging of the cells, and aspirating the media after letting the cells grow in the wells for 24 hours, then a master-mix of lipofectamine and GFP plasmid DNA was added to 500 microliters of stock DMEM, and then adding 100 microliters of the master-mix

1000 µg rat brain lysate was incubated with 50 µg construct overnight on a shaking incubator at 4°C. Samples were loaded on a polyacrylamide gel and processed for immunoblotting (see appendix) Immunoblotting for sacsin and JIP3 interaction Primary Sacsin and JIP3 antibodies (Santa Cruz cat. sc-515118 and sc-46663) raised in mice were added in a 1:3000 and 1:2000 ratio respectively to the corresponding membrane sections and incubated on a shaking incubator for 1 hour. Next, excess antibody was removed

dysbindin-1 binds to Drp1 to enhance its oligomerization. To test this possibility, we transfected HEK-293 cells with YFP-Drp1 along with HA-tagged dysbindin-1C or -1A, and used an anti-HA antibody to immunoprecipitate dysbindin-1 at 2 days after transfection. Drp1 was detected in the immunoprecipitation product from cells transfected with dysbindin-1C, but not with -1A plasmid (Figure 4G), indicating that

capability of Aβ clearance in AD, we suspected that miR34a may be an indispensable player. To validate this, Aβ42 fibrils were prepared from Aβ42 monomer ex vivo and used to treat glioma A172 and U251 cells. Aβ uptake was blocked by both transient transfection of a miR34a mimic and stable miR34a overexpression (Supplementary Fig. 1B, Fig. 1A), but enhanced by anti-miR34a (Fig. 1B), indicating that miR34a suppressed Aβ phagocytosis in these cells. The fact that enhanced and decreased levels of miR34a

Transfection of pCas9-GFP with HEK293 cells Introduction Transfection is the insertion of plasmid DNA into eukaryotic cells. Transfection is used to look at the expression of a gene inserted in a cell. For this laboratory, the eukaryotic cells used were HEK293 cells. 293 cells are derived from human embryonic kidney cells. The reagent used to transfect pCas9-GFP into 293 cells was polyethylenimine (PEI). PEI is a cationic polymer and is the transfecting agent used to introduce pCas9-GFP into the

the green fluorescent in helper virus, however, this process was more complex so we skipped this step. The next step that was extraction the plasmid from the bacteria by following a specific protocol and then started working with PEI Transfection.

The CCR5 co-receptor is not only the center of research with stem cell transplant, but has also been a recent target of gene therapy research. Gene therapy is a fairly new technology where genes (edited or normal) are transplanted in humans to produce a specific response. One avenue of research that is currently being studied is T cell gene editing focused on the CCR5 delta32 mutation in HIV infected patients using the CRISPR/cas9 system. There is some research using TALENS, but evidence with CRISPR/cas9

cavitation bubbles will disruptive the membrane of cells and tissues to enhance drug transport. Use the ultrasound to enhance cell permeability is a new methods for gene transfection. The increase uptake of DNA or RNA has been demonstrated by several investigators2-1, 2-5. However, the further enhancement of gene transfection

dendrimers are the most frequently used due to their high transfection efficiency. The transfection efficiency is highly influenced by the size of dendrimers and the type of amine groups that they are composed of. The primary amine groups participate in DNA binding, so they promote DNA cellular uptake. However, the tertiary amino groups reinforce the release of DNA into the cytoplasm because they act as a proto-sponge in endosomes. Transfection efficiency can be enhanced by increasing the size and diameter

The RNA interference (RNAi) pathway is an important biological system that is utilised by many organisms as a method of regulating gene expression. But what exactly is RNAi? And what is the importance of its use and application in biology? RNAi is a cellular process that actively silences specific genes. This method of post-translational gene regulation has been observed in many organisms including plants, fungi, some bacteria and animals. RNAi inhibits protein synthesis of the target transcript