Transfection in animal cells through chemical methods like Calcium phosphate precipitation,electroporation,microinjection,lipofection

Transfection in animal cells and it's methods

1. CENTRAL UNIVERSITY OF HARYANA COURSE NAME : Animal Biotechnology Topic : Transfection of animal cells – Calcium phosphate coprecipitation ,electroporation , lipofection, Peptide , direct DNA transfer , viral vectors,microinjection Presented to Dr. Ram Gopal Nitharwal Department of Biotechnology Presented by Smriti Ranjan Msc.Biotech (III sem ) Roll no. 221265

2. T ransfection is the process of introducing foreign genetic material, such as DNA or RNA, into eukaryotic cells. It is a crucial technique in molecular biology and biotechnology for studying gene function, protein expression, and genetic engineering. There are various methods for transfecting animal cells. Transfection , technique used to insert foreign  nucleic acid  ( DNA  or  RNA ) into a  cell , typically with the intention of altering the properties of the cell. The introduction of nucleic acid from a different cell type can be accomplished using various biological, chemical, or physical methods .

3. There are two type of transfection : STABLE TRANSFECTION TRANSIENT TRANSFECTION In stable transfection , the plasmid DNA successfully integrates into the cellular genome and will be passed on to the future generation of the cells. In transient transfection , the transfected material enters the cell but does not get integrated into the cellular genome . Thus , a transiently transfected cell will only express transfected DNA for a short amount of time and not pass it on to daughter cells.

4. This methods are divided into 3 categories : Chemical methods Calcium Phosphate Lipids Cationic polymer 2. Physical methods Electroporation Microinjection Laserfection Sonoporation Bioloistic particle deliivery 3.Biological methods Virus-based

5. CALCIUM PHOSPHATE TRANSFECTION The calcium phosphate transfection technique involves the precipitation of DNA and calcium phosphate. The precipitation is facilitated by mixing a HEPES-buffered saline solution, having sodium phosphate, with calcium chloride solution and DNA. Glycerol shock is often used to enhance the DNA uptake in certain cells. While this technique is cost-efficient and can be used for transient or stable transfections in a wide range of cells, relatively small changes in pH (±0.1) can affect the efficiency of transformation. Furthermore, it is essential to maintain reagent consistency for reproducing the assay results. However, this transfection method does not work in RPMI, or other media with high phosphate concentrations.

6. Figure : Mechanisms of chemical transfection, including calcium phosphate precipitation.  In calcium phosphate precipitation specifically, a calcium-phosphate-DNA co-precipitate is formed which facilitates binding to the cell surface and entry of the nucleic acid into the cell via endocytosis . In the calcium phosphate precipitation method, the calcium phosphate facilitates the binding of the condensed DNA in the co-precipitate to the cell surface, and the DNA enters the cell by endocytosis. Aeration of the phosphate buffer while adding the DNA-calcium chloride solution helps ensure the precipitate that forms is as fine as possible, which is important because clumped DNA will not adhere to or enter the cell as efficiently.

7. Advantages: Relatively simple and inexpensive. Suitable for a wide range of cell types. Disadvantages: Variable efficiency. Cellular toxicity due to the precipitation of calcium phosphate.

8. LIPOSOME-MEDIATED TRANSFECTION Liposome-mediated transfection (lipofection) techniques involve the use of liposome forming cationic lipids, or non-lipid polymers. Examples of lipofection transfection reagents may include DOTMA (N-[1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)and X- tremeGENE ™ transfection reagents, suitable for transfecting a variety of DNA, small RNA, and CRISPR/Cas9 components into a diverse range of cell lines. Lipid transfections can also be adapted for cost-effective, as well as high-throughput systems ; however, these transfections are usually cell-type specific.

9. Advantages: Versatile and effective for a variety of cell types. Low cytotoxicity compared to some other methods. Disadvantages: Batch-to-batch variability. Limited cargo capacity for large DNA fragments.

10. ELECTROPORATION This technique involves the exposure of cell membranes to high-intensity electric pulses which causes a temporary destabilization in certain areas of the cell. During this transient destabilization event, the cell membrane becomes highly permeable and allows the entry of various exogenous molecules including DNA 4 . Electroporation is an easy, non-chemical technique that can yield high transformation efficiencies in various cell types. Although this technique does not alter target cell morphology and functions, the method can cause cell death if transfection is not performed under optimum conditions.

11. Electroporation, also called electropermeabilization , is an efficient, non-viral delivery system that allows genetic material (DNA and RNA), proteins, drugs or other molecules to enter cells. It uses an accurately pulsed electrical current to create temporary pores in the cell membrane through which the molecules can then pass. This process can be used on a wide variety of cells including mammalian, 1  insect, 2  yeast, 3  plant 4  and bacterial cells . 5 Fig : Schematic diagram showing the steps and associated charges during electroporation that lead to the introduction of exogenous material into the cell, in this case a plasmid.

12. Advantages: High efficiency. Suitable for a broad range of cell types. Disadvantages: Cell viability can be compromised. Requires specialized equipment.

13. VIRAL TRANSFECTION (VIRAL TRANSDUCTION) This method involves the use of viral vectors to deliver nucleic acids into cells. Viral delivery systems such as lentiviral, adenoviral and oncoretroviral vectors can be used for transferring nucleic acids, even in hard-to-transfect cells. Although viral delivery methods are highly efficient, they can be quite laborious. Moreover, most viruses require containment and careful monitoring of biosafety levels. Before performing viral transfections, it is also important to consider several limiting factors such as the lytic nature of viral vectors, cell line packaging and host-cell specificity

14. https://www.sciencedirect.com/science/article/pii/S0753332220304686

15. Peptide transfection is a method used to deliver nucleic acids (such as DNA, RNA, or siRNA) into cells using cationic peptides or protein transduction domains (PTDs). This approach is an alternative to traditional transfection methods like lipofection or electroporation. Peptide transfection has gained attention due to its relative simplicity, low cytotoxicity, and potential for targeted delivery. Peptide

16. Mechanism of Peptide Transfection: Formation of Peptide-Nucleic Acid Complex: Cationic peptides interact with the negatively charged nucleic acids, forming a complex. This interaction is often driven by electrostatic forces. Cellular Uptake: The positively charged peptide-nucleic acid complex interacts with the negatively charged cell membrane. Various mechanisms, such as endocytosis or direct translocation, may be involved in the internalization of the complex into the cell. Endosomal Escape: If endocytosis is involved, the complex may be encapsulated in endosomes. To express the delivered genetic material, it needs to escape from the endosomes into the cytoplasm. Some peptides possess endosome-disruptive properties, facilitating the release of the cargo into the cytoplasm. Transport to the Nucleus (if applicable): If the delivered material is DNA, it needs to reach the nucleus for gene expression. Some peptides may have nuclear localization signals or facilitate transport to the nucleus. Expression of Transferred Genetic Material: Once in the cytoplasm or nucleus, the delivered nucleic acid can be transcribed and translated, leading to the expression of the encoded protein or other biological effects.

17. Advantages of Peptide Transfection: Low Cytotoxicity: Peptide transfection is often considered less toxic to cells compared to some other transfection methods. Targeted Delivery: Some peptides can be modified to have cell-type or tissue-specific targeting properties. Ease of Use: The simplicity of the method makes it attractive for certain applications. Challenges and Considerations Efficiency: Peptide transfection may have lower efficiency compared to other methods, particularly in certain cell types. Cargo Size Limitations: The size of the nucleic acid cargo that can be effectively delivered may be limited. Optimization: Optimization of peptide sequences and experimental conditions may be required for different cell types and applications. Endosomal Entrapment: Efficient endosomal escape can be crucial for successful transfection, and some peptides may require modifications to enhance this process.

18. Direct DNA transfer is a method of introducing foreign DNA into cells without the use of carriers such as viruses, liposomes, or peptides. This technique is often used in research settings, and it is relatively simple compared to other transfection methods. However, direct DNA transfer is generally less efficient than some other methods, and its success can depend on various factors such as cell type, DNA size, and the method of delivery. Direct DNA transfer

19. Methods of Direct DNA Transfer: Microinjection: In microinjection, a fine micropipette is used to physically inject DNA directly into the nucleus or cytoplasm of a cell. This method is highly precise but is labor-intensive and not suitable for high-throughput applications. Particle Bombardment (Gene Gun): In particle bombardment, gold or tungsten particles coated with DNA are shot into target cells using a gene gun. The particles penetrate the cell membrane, delivering the DNA directly into the cell.

20. Mechanism of Direct DNA Transfer: Microinjection: Cell Selection: Cells are typically cultured and selected based on their adherence and health. Micropipette Preparation: A micropipette is filled with the DNA solution. Cell Microinjection: The micropipette is carefully inserted into the target cell, and the DNA solution is injected directly into the nucleus or cytoplasm. Cell Recovery: The injected cells are allowed to recover, and their subsequent behavior is observed.

21. Particle Bombardment (Gene Gun): Preparation of DNA-Coated Particles: DNA is coated onto gold or tungsten particles. Cell Preparation: Target cells are typically placed on a solid support, such as a petri dish. Particle Bombardment: The DNA-coated particles are accelerated and shot into the target cells using a gene gun. Cell Recovery: The cells are allowed to recover, and the expression of the introduced DNA is monitored.

22. Advantages of Direct DNA Transfer: No Need for Carriers: Direct DNA transfer eliminates the need for viral vectors, liposomes, or other carriers. Simple Procedure: Microinjection and particle bombardment are relatively simple techniques, requiring basic laboratory equipment. High Precision: Microinjection allows for precise control over the amount of DNA injected into each cell. Challenges and Considerations: Cell Viability: The physical nature of direct DNA transfer methods can affect cell viability, and optimization is required for different cell types. Efficiency: Direct DNA transfer methods are generally less efficient compared to some other transfection methods. Cell-Type Specificity: The success of direct DNA transfer can vary depending on the cell type. Potential Cellular Damage: Microinjection can potentially cause cellular damage, and care must be taken to minimize this.

23. https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/calcium-phosphate-precipitation.html References