Table of Contents
Transfection is the processes in which an exogenous genetic material such as DNA or RNA is introduced into a cell (Chapman et al., 2013, p.93). Such a process may be achieved through a physical, chemical, or biological approach. The impact of transfection is that the introduced genetic material can elicit the associated genetic trait in the recipient cell as well as the possibility becoming incorporated into the host cell’s genome (Chapman et al., 2013, p.93).
Calcium Phosphate Transfection
As the name suggests, calcium phosphate transfection is a chemical based process and one that is among the least expensive methods (Sun et al., 2013; Kaestner, Scholz & Lipp, 2015). The origin of the method is dated back in the early 1970s and associated with F. L. Graham and A. J. van der Eb (Kaestner, Scholz & Lipp, 2015). In the method A HEPES-buffered solution that is a blend of the genetic material and calcium chloride as well as phosphate ions. The chemical reaction between calcium and phosphate ions ends up with the formation of a precipitate upon which the genetic material that is to be transfected is bound (Mostaghaci, Loretz, and Lehr, 2016). The precipitate-genetic material complex is then introduced into the recipient monolayer cell cultures and it happens that as the cells absorb the precipitate they also absorb the genetic material. The transfected genetic material is then able to express its associated genetic trait in the recipient cell and can also become incorporated into the genome of the recipient cell. As a way of increasing the efficiency of the transfection process, glycerol or DMSO is always added to the recipient cells layer (Kingston, Chen, and Rose, 2003).
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Materials and Reagents Used In a Calcium Phosphate Transfection Experiment
The materials and reagents used in the Calcium Phosphate Transfection experiment as well as their specific quantities and parameters are indicated in the table below according to Bingnan (2014)
|Materials/ Reagents/ Parameter||Specific Quantity|
|Relative Surface Area||58.1 cm2|
|Culture Media||10 ml|
|Chloriquine 25mM||10 µL/well|
|HeBS buffer||600 ul|
|2.5M CaCl||60 ul|
|Transfection Media||1200 ml|
|Total DNA||20 ug|
Table 1: Materials and Reagents Used In a Calcium Phosphate Transfection Experiment
Procedure of a Calcium Phosphate Transfection Experiment
- On the day of transfection, four replicates of the DNA mix are prepared in the absence of CaCl2 and H2O.
- The samples are then thawed and the HeBS buffer pH adjusted to 7.05.
- The transfection buffer is then sterilized with a 0.22µm filter.
- H2O and CaCl2 are then added to each tube.
- Chloriquine is then added to the wells/plates with the cells transfected within 5 to 10 minutes after chloriquine is added.
- The CaCl2-DNA complex is added methodically to the HeBS buffer through a mechanical bubbling process using a 1 ml pipette. The process is in such a way that as bubbling is done in the HeBS buffer, the CaCl2-DNA complex is added drop by drop to facilitate the formation of small precipitates.
- The resulting mixture is then put into the media plate and shaken gently to blend with mix with the media.
- The mixture is then incubated for 5-7 hours at 37 ºC and 5% CO2
- The cells are washed once using 1X PBS then aspirated and fresh 10% FBS-DMEM media added.
- The 10 cm plate cells are harvested after 48 hours for biochemical assessment.
- Bingnan, (2014). Calcium phosphate transfection.
- Chapman, H.D., Barta, J.R., Blake, D., Gruber, A., Jenkins, M., Smith, N.C., Suo, X. and Tomley, F.M., (2013). A selective review of advances in coccidiosis research. Adv Parasitol, 83(354), pp.93-171.
- Kaestner, L., Scholz, A. and Lipp, P., (2015). Conceptual and technical aspects of transfection and gene delivery. Bioorganic & medicinal chemistry letters, 25(6), pp.1171-1176.
- Kingston, R.E., Chen, C.A. and Rose, J.K., (2003). Calcium phosphate transfection. Current protocols in molecular biology, pp.9-1.
- Mostaghaci, B., Loretz, B. and Lehr, C.M., (2016). Calcium phosphate system for gene delivery: Historical background and emerging opportunities. Current pharmaceutical design, 22(11), pp.1529-1533.
- Sun, M., Bernard, L.P., DiBona, V.L., Wu, Q. and Zhang, H., (2013). Calcium phosphate transfection of primary hippocampal neurons. Journal of visualized experiments: JoVE, (81).