The document summarizes various methods for transfecting animal cells, including calcium phosphate coprecipitation, lipofection, electroporation, viral vectors, microinjection, and direct DNA transfer. It provides details on the mechanisms and advantages/disadvantages of each method. Calcium phosphate transfection involves precipitating DNA with calcium phosphate to facilitate cell binding and entry. Lipofection uses cationic lipids to form complexes with nucleic acids for cellular uptake. Electroporation exposes cells to electric pulses to temporarily destabilize membranes and allow DNA entry. Viral vectors are highly efficient but require biosafety precautions. Microinjection and gene guns directly inject or bombard DNA into cells.

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)

2. Transfection 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 :
1. Chemical methods
Calcium Phosphate
Lipids
Cationic polymer
2. Physical methods
Electroporation
Microinjection
Laserfection
Sonoporation
Bioloistic particle deliivery
3.Biological methods
Virus-based

7. 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.

8. 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.

10. •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.

11. 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.

12. •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.

13. 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 DNA4.
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.

14. 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 plant4 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.

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

16. 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

17. https://www.sciencedirect.com/science/article/pii/S
0753332220304686

18. 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

19. Mechanism of Peptide Transfection:
1.Formation of Peptide-Nucleic Acid Complex:
1. Cationic peptides interact with the negatively charged nucleic acids, forming a complex.
This interaction is often driven by electrostatic forces.
2.Cellular Uptake:
1. The positively charged peptide-nucleic acid complex interacts with the negatively charged
cell membrane.
2. Various mechanisms, such as endocytosis or direct translocation, may be involved in the
internalization of the complex into the cell.
3.Endosomal Escape:
1. 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.
2. Some peptides possess endosome-disruptive properties, facilitating the release of the
cargo into the cytoplasm.
4.Transport to the Nucleus (if applicable):
1. 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.
5.Expression of Transferred Genetic Material:
1. 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.

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

21. 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

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

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

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

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

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