Lipo2000 Transfection Reagent

Introduction

It is a type of reagent that is cationic, liposome-based, and used in a wide variety of in-vitro techniques in mammalian cells for transfection, and transgene expressions.  The efficiency of transfection and viability of cell depends upon many factors which are given as follows:

• Media components (antibiotics & serum level)
• Liposome
• Cell density
• DNA-liposome complexing time
• DNA concentration

For a transfection to be successful, there must be a normal physiological interaction between the nucleic acid and the cell membrane because both of them carry a negative charge. As we have discussed that Lipo2000 is cationic, so it functions by making a complex with the nucleic acid and allows its interaction with the cell membrane to overcome its repulsive electrostatic forces and move into the cell. So these lipid molecules due to their structural variations (due to the saturation and cis-trans configuration) easily mediate the process of transfection because of having some common features(Gao and Huang, 1995). First, all the lipid molecules have one or more positively charged nitrogen atoms in the head groups that allow them to interact with the negative charged nucleic acid and the transfection reagent. This spacer has one or more chains of hydrocarbons that are linked to the head region. Also, these spacers play a major role in the interaction between the nucleic acid and lipid molecules which is cationic. These cationic lipid molecules are formed with the use of helper lipids. Ideally, it is formed by mixing two lipid molecules which by the process of sonification converted into uniform size liposomes (almost 100nm). These helper lipids allow the fusion of liposomes with negatively charged nucleic acid and mediate the entry. To get the transgene expressions, there must be the interaction of DNA into the nucleus of the cell so that it can get access to the transcription process. This DNA which by transfection get into the nucleus, and then there will be the reassembly of the nuclear envelope within the mitotically dividing cell(Zabner et al., 1995, Zuhorn et al., 2002). However, it can also promote the transfect into the rat’s hepatocytes and post-mitotic cells.

General considerations for transfection

Cell density

 Generally, cell density should be 90-95 % to lessen the drop in cellular growth. Studies indicate that any type of variation within the cell density during the process of transfection leads to variation in gene expression. If the cell density increases, it will cause a decrease in the growth and metabolism of cells. And if it decreases, it will affect the recovery of culture.

 DNA concentration and Lipofectamine 2000 and DNA ratio

Lipo2000 reagent is available in the concentration of 1mg/ml and the ratio of Lipo2000 reagent to DNA should be 2:1 or 3:1. If the DNA is available in a high concentration, a higher ratio can be used. For example, 0.8 to 1.0 microgram of plasmid DNA along with the 2-3 microliters of Lipo2000 reagent can be helpful. When this ratio is optimized according to the available concentration of DNA, then DNA must be in sufficient concentration to allow effective transgene expressions.

Dilution time for Lipo2000 transfection reagent

Before the mixing of Lipo2000 with DNA, the Lipo2000 reagent must be diluted in OOP-MEM I. According to the volume of the flask, it should be diluted as 25-fold or 50-fold. Studies show that the dilution time between the reagent and DNA affects the transfection activity of Lipo2000. After dilution, it should be incubated for 5 minutes. To ensure the peak or effective transfection efficiency, it should be diluted for almost 20 to 30 minutes. If there is no complex formation between lipid and DNA within 60 to 90 minutes of dilution, then there will be a decrease in transfection efficiency.

Lipo2000 and DNA complexing

Upon mixing of Lipo2000 reagent and diluted DNA, there will be no immediate formation of complexes. To figure out the best complex formation time between DNA and Lipo2000, CHO-S cells are transfected using an enzyme called beta-galactosidase. Results show that the expression for beta-galactosidase increases as the time for complex formation increases. The optimal time for complexing is 30 minutes. As the time increases from 30 minutes, the activity of either DNA or Lipo2000 reagent decreases. But these complexes are stable enough that they can provide greater expression after 2 hours of mixing or sometimes, even after 6 hours of mixing.

Conditions related to cell culture

Studies related to the conditions of cell culture are not described. Although it is described that the health of a cell is an important tool for variability from one to another transfection process. For example, after the thawing process of culture stock, it should not be maintained for more than 30 passages. As the passage time increases, it will drastically affect the efficiency of transfection along with the transgene expression. Culture cells must have the ability to grow well and this will surely affect the transfection efficiency.

Applications

There are many applications in which Lipo2000 reagent can be used. Some of these applications are described below:

Transfection into the siRNAs by Lipo2000 reagent

For RNAi studies, Lipo2000 transfection reagent is used to transfect the short interfering RNAs (which are produced synthetically) into the types of different mammalian cells. Many factors can influence the target genes in the RNAi experiments. These includes

• Mammalian cell itself and its growth ability
• Protein stability
• Rate of transcription for the gene of interest
• mRNA stability
• Transfection efficiency

 Primary neuron transfection by using lipofectamine 2000

 The transfection ability into the primary cells is somewhat problematic and has low efficacy rates.  The improvements which are done in the recent era allow us to study different processes including neuron physiology and its pathophysiology along with neurogenesis.

HTP transfection

It is also called a high throughput transfection. It is done in the 96 or 364 well plate format. Scientists have also developed different protocols in which they use Lipo2000 reagent for transfection purposes(Dalby et al., 2004).