What methods are used to transfect mammalian cells?
The methods include direct micro injection, biolistic particle delivery, electroporation, and laser-based transfection [13]. In brief, the micro injection method directly injects nucleic acid into the cytoplasm or nucleus [14, 15].
What are U937 cells used for?
U-937 cells are a model cell line used in biomedical research. They were isolated from the histiocytic lymphoma of a 37-year-old male patient and are used to study the behaviour and differentiation of monocytes.
How do you stably transfect a cell line?
One of the most reliable ways to select cells that stably express transfected DNA is to include a selectable marker on the DNA construct used for transfection or on a separate vector that is co-transfected into the cell, and then apply the appropriate selective pressure to the cells after a short recovery period.
What are the two types of transfection?
Generally, transfection can be classified into two types, namely stable and transient transfection (Kim & Eberwine, 2010; Stepanenko & Heng, 2017).
What are the different methods of transfection?
Transfection Methods
- Transient Transfection.
- Stable Transfection.
- Cotransfection.
- Electroporation.
- Cationic Lipid Transfection.
What is the principle of transfection?
The Principle of Transfection
Transfection is defined as the process of inserting nucleic acids (e.g., plasmid DNA, cDNA, mRNA, miRNA, siRNA) into the cytoplasm of eukaryotic cells. In addition, proteins and nanoparticles such as beads or dyes can be transfected.
What type of cells are U937?
U937 is a pro-monocytic, human myeloid leukaemia cell line and was isolated from the histiocytic lymphoma of a 37 year old male (Sundstrom and Nilsson 1976). This cell line exhibits many characteristics of monocytes and is easy to use.
Is U937 an AML cell line?
The U937 cell line was originally established from the pleural effusion of a patient with histiocytic lymphoma and is usually considered to be an AML cell line (10, 11).
What does stably transfected mean?
Stable transfection refers either to the permanent expression of the gene of interest through the integration of the transfected DNA into the nuclear genome, or the maintenance of a transfected plasmid as an extra chromosomal replicating episome within the cell.
What is the difference between transient and stable transfection?
In transient systems, foreign DNA, unable to replicate independently from the host’s DNA, persists only for a few days. In contrast, with stable transfection, foreign DNA is integrated into the genome, replicated alongside it, and, more importantly, passed down to the progeny.
What are the methods of cell transfection?
What are the steps of transfection?
Chemical-mediated transfection
- encapsulation of genetic material with transfection reagent.
- Cellular uptake of nanoparticles.
- Release into the cytosol and if needed transport into the nucleus for transcription.
What is the process of transfection?
Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment.
Are U937 cells adherent?
Culture of U937 Cells
U937 cells were differentiated by culture at a starting cell concentration of 1 × 106 cells/ml in medium containing 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 hr. The cells were then adherent and somewhat flattened.
How do you know if a transfection was successful?
Determining the number of positive cells within a transfected cell population can be done through microscopy and flow cytometry. Finally, confirming localization of your protein of interest can be done by microscopy.
What are the methods of transfection?
What are advantages of stable transfection over transient transfection?
In stable transfection, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell. However, in transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome.
What is chemical method of transfection?
The most commonly used method in clinical trials is the biological method where transfected nucleic acids are delivered to cells by viruses. The chemical transfection methods are techniques that catalyze DNA cross-membrane transport through the use of Ca2+phosphate, polycations or dendrimers.
What is the difference between transformation and transfection?
The main difference between transfection and transformation is that the transfection refers to the introduction of foreign DNA into mammalian cells while the transformation refers to the introduction of foreign DNA into bacterial, yeast or plant cells.
What enzyme is used during the process of transfection?
Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, electroporation) are used in order to fuse the protoplast carrying the gene of interest with the target recipient cell.
Why do we transfect cells?
The two main purposes of transfection are to produce recombinant proteins, or to specifically enhance or inhibit gene expression in transfected cells.
What factors affect transfection efficiency?
Factors Influencing Transfection Efficiency. With any transfection reagent or method, cell health, degree of confluency, number of passages, contamination, and DNA quality and quantity are important parameters that can greatly influence transfection efficiency.
What is a good transfection efficiency?
The viability and general health of cells prior to transfection is known to be an important source of variability from one transfection to another. In general, cells should be at least 90% viable prior to transfection and have had sufficient time to recover from passaging.
How much DNA is needed to transfect?
In general, use 1 µg of DNA per 1 ml of culture to be transfected. PEI and DNA should each be diluted into 1/20 of the total culture volume before being combined.
How can you increase the efficiency of a transfection?
Improving the Success of Your Transfection
- Transfect healthy, actively dividing cells at a consistent cell density.
- Transfect using high-quality DNA.
- Optimize the amount of DNA used to transfect cells.
- Optimize the transfection reagent:DNA ratio.
- Optimize cell number per well when transfected.