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What is the difference between transformation and transfection?

What is the difference between transformation and transfection?


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What is the difference between transformation and transfection? How do both of these methods work?


If you are interested in the history of molecular biology this is an interesting question.

Basically transformation came to be used to describe experiments in which the phenotype of an organism was changed by the uptake of DNA, and because of the way this developed in bacterial systems this DNA was usually a plasmid. Then it became possible to use purified phage DNA to infect cells, whereupon the 'transformed' cells produced phage particles - for obvious reasons this was called transfection.

When efforts turned to getting the same techniques working with cultured animal cells many of the vectors were based on viral genomes (e.g. SV40) and these workers referred to this as transfection. At this point the distinction between the two terms became meaningless, and usage tends to be for historical reasons. Interestingly in molecular manipulations of yeast, where there are no viruses, the transfection word is rarely used.


As far as I know (and I haven't found no evidence against it) this is mostly a semantic difference. Both processes describe the addition of genetic material into cells using various techniques.

Transformation is here mostly used for bacterial work (transforming plasmids for example), while transfection is almost exclusively used for eukaryotic cells. The reason may be, that the term transformation is used in eukaryotic cells as well to describe the progresion of cells into cancer cells.

The techniques are not very different and there are a lot of them. Transfections often use transfection agents, which form pores in the cell membrane through which the DNA can enter the cell. This can also be done by using a short electropulse. A lot of transformations are done using "chemically treated" cells, where the cells are brought into a state in which they take up DNA which adheres to their outside.

The Wikipedia articles on Transfection and Transformation gives you more details on the methods.


What Are Transformation, Transfection & Transduction?

One of the pillars of modern day molecular biology uses techniques to manipulate DNA sequences (such as plasmids, knockout gene constructs, etc.) and introduce them into a host cell to test their effects. However, getting the DNA into cells can take different routes. Those unfamiliar with the field may be wondering “what is plasmid transduction?” Or have heard the terms transformation, transfection, and transduction, but are uncertain as to the differences and similarities between these techniques. Although these terms have some overlap, and so their usage is often confusing or incorrect.

What Is Plasmid Transformation?

Transformation is, simply put, the process of altering a cell’s genetic code through the uptake of foreign DNA from the environment. Plasmid transformation is used to describe the (non-viral) horizontal gene transfer of plasmids between bacteria. While transformation likely happens in the natural world, scientists have harnessed this process to their own ends, enabling replication of lab-manipulated plasmids and expression of desired recombinant DNA sequences.

The process is relatively simple scientists make the membranes of bacterial cells permeable to DNA either through chemical means or via electrical stimulation. These cells, now termed ‘competent cells,’ will readily uptake plasmid DNA from their surroundings. Once the DNA molecule of interest is introduced to these competent cells, the bacteria have now been plasmid transformed. The transformed cells then can be selected from the untransformed cells by inclusion of an antibiotic to kill off the untransformed cells. Typically, this occurs as the plasmid will express an antibiotic resistance gene to protect the transformed cells and ensure maintenance of the plasmid over time and cell divisions. In the process, many replicons of the plasmid will be created and passed to daughter cells.

What Is Plasmid Transfection?

Transfection is a type of plasmid transformation, typically that of animal cells, instead of bacteria. This process is a bit more complicated than your run-of-the-mill transformation, as many lab-cultured eukaryotic cells do not natively uptake and replicate foreign DNA. Still, scientists have discovered many ways in which plasmids and other foreign DNA can be introduced to cells.

Much like methods for bacteria, there are both chemical and physical methods of transfection produce transient holes in the cell membrane and get uptake of foreign DNA. These methods work similarly to the those outlined for bacterial transformation, as they all are designed to make the cell membrane more permeable. The method by which they do so is different from bacteria, though, instead using cationic lipids, micelles, lasers, or even particle guns. These methods have their pros and cons, but ultimately will depend on the resources available and the preference of the researcher.

What Is Transduction?

The final prominent method, transduction, is unique from the other two methods. Transduction is the process of using a virus to mediate the delivery of DNA fragments or plasmids into a cell, either prokaryotic or eukaryotic. This technique harnesses the natural function of viruses to inject DNA into the infected host, but with a twist. Scientists can modify the viral nucleic acids to contain specific DNA sequences of interest. There are many different types of viruses that can be manipulated to introduce recombinant nucleic acids into host cells. For example, bacteriophage introduce DNA into bacteria, and lentiviruses or adenoviruses into human cells. Using these modified viruses, researchers incorporate foreign DNA into the host genome (such as using lentiviruses or bacteriophage) or transiently express desired recombinant nucleic acids (such as using adenoviruses).

In order to perform a transduction, you need a cell-line of interest and a virus that infects that cell line. This method can be more difficult than the other methods discussed here, since the virus must be grown and maintained in culture, sometimes needs to be modified to be non-infectious to humans, and the DNA of interest must be packaged into the viral particle before infection of new host cells can occur. Despite the challenges to overcome, viral transduction is an excellent way to perform stable, long term transformations and transfections in the lab environment.


Transient transfection

In transient transfection, the introduced nucleic acid exists in the cell only for a limited period of time and is not integrated into the genome. As such, transiently transfected genetic material is not passed from generation to generation during cell division, and it can be lost by environmental factors or diluted out during cell division. However, the high copy number of the transfected genetic material leads to high levels of expressed protein within the period that it exists in the cell.

Depending on the construct used, transiently expressed transgene can generally be detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to 96 hours post-transfection. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The optimal time interval depends on the cell type, research goals, and specific expression characteristics of the introduced gene, as well as the time it takes for the reporter to reach steady state. However, within a few days most of the foreign DNA is degraded by nucleases or diluted by cell division after a week, its presence is no longer detected. Transient transfection is most efficient when supercoiled plasmid DNA is used, presumably due to its more efficient uptake by the cell. siRNAs, miRNAs, mRNAs, and even proteins can be also used for transient transfection, but as with plasmid DNA, these macromolecules need to be of high quality and also be relatively pure (see Factors Influencing Transfection Efficiency). While transfected DNA is translocated into the nucleus for transcription, transfected RNA remains in the cytosol, where it is expressed within minutes after transfection (mRNA) or bound to mRNA to silence the expression of a target gene (siRNA and miRNA) (see Guidelines for RNA Transfection).


Replicate controls: technical and biological

Reliable results are reproducible from properly controlled experiments. The observed phenotype associated with the independent variable should be consistent over time and from different preparations of the purified test plasmid expressing the independent variable. There are two types of replicate controls: technical and biological.

In general, technical replicates can be thought of as "plate controls" . They are NOT independent and are typically derived from one source. Conversely, biological replicates ARE independent and can be thought of as "reproducibility controls". Biological replicates are what makes up your sample size (aka your n value)" and should come from multiple, independent sources. We'll describe these a bit more below, but the papers cited in the reference section provide more in depth information.

Technical replicates

An example of a technical control is the transfection of multiple separate wells (within the same plate) with purified plasmid from the same aliquot/preparation. The replicate control isn’t measuring or assessing reproducibility of the effect of the independent variable because the purified plasmid, cells, and media used in each of the wells are not independent: they were derived from the same source and were incubated on the same plate at the same time. Although the replicate control is important, it speaks to the consistency of the reagents and hands performing the experiment, not the reproducibility or consistency of the observed phenotype associated with the independent variable.

Biological replicates

In any given set of experiments, the biological controls are more time consuming and ultimately more important. Ideally, each biological replicate should use fresh media, test independent aliquots of cells and plasmids, be performed on different days, etc, but outside constraints may impose some limitations and you should be cognizant of these when interpreting your results. The key to biological controls is independence: at the very least you should repeat your whole experiment from start to finish multiple times and on different days. Using our example above, we would test different preparations of Plasmid A in different aliquots of cells on several different days. Biological replicates control for the reproducibility and consistency of the observed phenotype associated with the independent variable and helps ensure the phenotype isn’t singular or associated with only one aliquot/preparation of the test plasmid.

Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection conditions. Now that we have successfully transfected our cells (Figure 3), we can continue with our experiment, incorporating additional positive and negative controls, performing multiple replicates, and ultimately getting interpretable results:

Figure 4: Expression Level of Gene X

The design and selection of proper experimental controls is not a trivial endeavor, as biological systems have many variables. However daunting designing and executing these steps may be, proper controls are a basic tenet of responsible scientific inquiry and investigation.

1. The problem of pseudoreplication in neuroscientific studies: is it affecting your analysis? Lazic SE. BMC Neurosci. Jan 1411:5. ( 2010) PubMed PMID: 20074371. PubMed Central PMCID: PMC2817684.

2. Replicates and repeats—what is the difference and is it significant? Vaux DL, et al. EMBO Rep. Apr 13(4): 291–296. ( 2012). PubMed Central PMCID: PMC3321166.


Both transfection and transduction can lead to a transient or stable expression of DNA into cells, depending on the method or the viral tool.

If a stable expression is required to maintain the foreign nucleic acid sequence in the genome of the cells and its daughter cells, then there are two options:

  • Co-transfect cells with a marker gene, which gives the cell some selectable advantage, such as resistance or labeling
  • Use a lentiviral vector to transduce target cells which naturally integrates its DNA into the host cell genome in a random manner.

On the other hand, transfection or transduction of RNA is always transient.

Transfection is efficient on adherent immortalized cells but primary and stem cells require transduction.

A limitation of the transfection approach lies in the toxicity of transfection for delicate cells, and its suspected effect on the expression of other genes or proteins.

For more information on the comparison between those two techniques and the bases necessary to know lentiviral vectors better, you can visit our Lentiviral vectors essentials page.


Gene Editing in Regenerative Medicine

Delivery Methods

Microinjection, gene gun , electroporation, and hydrodynamic delivery are all methods that have been used to deliver genome editing tools into cells [35–38] . These methods are not widely used because they either need specialized equipment and experienced professionals (microinjection) or they are associated with cell or tissue damage (gene gun, electroporation, and hydrodynamic delivery). These methods are also restricted in their ability to reach host tissues in vivo.

Chemical agents such as cationic lipids, polymers, and dendrimers have been widely used for gene delivery, but they are associated with gene delivery efficiencies that are generally lower than those of viral vectors. However, chemical delivery methods are still preferred compared with viral methods in some cases owing to their ease of preparation, lower immunogenicity, and lower expense [39] .

All of the chemical gene delivery methods can be applied to genome editing because there is no chemical difference between plasmids encoding targetable nucleases and plasmids encoding other genes. However, different mechanisms may be involved for the delivery of proteins. Cationic liposomes have been used to this end with relative success. The delivery of Cas9–gRNA complexes or TALENs via cationic lipids is evident by the multiple commercially available agents that are in the marketplace [40,41] . The cationic polymer poly(ethylenimine) has been used to coat self-assembled DNA cages to delivery Cas9/gRNA complexes successfully into mammalian cells [42] . Cell-penetrating peptides have been used to deliver targetable nuclease proteins, including Cas9 and TALEN [43,44] . Interestingly, ZFN has inherent cell-penetrating capabilities and can pass into cells without a carrier [45] .

Viruses have been used for gene delivery for decades [46] . For gene editing applications, several viruses have been evaluated, including lentivirus [47] , adenovirus [48] , and adeno-associated virus (AAV) [49] . Lentiviral vectors have been shown to have a high transduction efficiency in multiple primary cell types and are capable of having genes integrated into the genome of the host [39,50] . As mentioned, incorporating a gene encoding a nuclease into the genome can lead to constitutive expression of the enzyme, which can lead to off-target events. Even worse, lentiviral integration occurs randomly, which can potentially induce various malignancies [51,52] . One solution to these problems of integration is to knock out the viral gene that encodes integrase, a technique that has been used to transfer genes encoding ZFNs, TALENs, and Cas9 [20,53] . Another solution is to develop lentivirus-derived particles to carry nuclease proteins. ZFN, TALEN, and Cas9 proteins have been successfully packed into such lentiviral particles [47,54] . Donor templates have been copacked into the particles to achieve targeted DNA insertion and gene correction [54] . It has been reported that this approach has lower off-target activity than traditional delivery methods [47,54] .

Adenoviral vectors allow transient transgene expression in both dividing and nondividing cells. These vectors carry a much lower risk of genomic integration than their retroviral counterparts. A set of third-generation adenoviral vectors named “helper-dependent adenoviral vectors” have increased cargo sizes (36 kb, as opposed to ∼8 kbp) [55] , which provides more versatility for the delivery of donor DNA templates for gene insertion and correction. However, immunogenicity is still a concern, as it is for all virus methods [56] , especially when multiple gene delivery events must be carried out.

Like adenovirus, AAV can produce transient transgene expression in both dividing and nondividing cells. In gene delivery, certain AAV serotypes are preferred because their delivered genes will be integrated into the host genome in a targeted fashion. Keeping in mind the previous discussion about problems with constitutively expressed nucleases, recombinant AAV have been produced that lack the viral rep gene to lower or eliminate the frequency of genomic integration [57] . Some strains of AAV carry lower immunogenicity concerns for a single administration, but repeated administrations require different serotypes to avoid a secondary immune response. These viruses are relatively small so they have limited DNA loading capacity (∼5 kb) [58] . The ZFNs with double monomers [59] are usually compatible with this limitation, but it is difficult to package double TALEN monomers (∼3 kb each) or SpCas9 (∼4.2 kb) and associated gRNA within a single AAV vector [60] . For in vitro genome editing, this limitation can be overcome by packaging the functional units separately and cotransducing cells. Edited cells can then be screened and selected. However, this is not practical for in vivo editing because the selection step cannot be performed, and every abnormal cell is expected to be treated. One could reduce the size of the gene encoding the enzyme: a Cas9 orthologue, named S. aureus Cas9 (SaCas9), which is 1 kb smaller than SpCas9, was identified [60] . The smaller gene has been successfully copacked with gRNA in a single AAV construct, with precise genome editing still being attainable.


The Pros and Cons of Each

Electroporation is less cumbersome than chemical transformation and generally gives higher transformation efficiencies (measured in colonies formed per microgram of DNA). However, it is more expensive. It requires a specialized apparatus to deliver the charge and cuvettes to transfer the charge to the cell suspension. Electroporation is sensitive to salt – you can lose precious samples if excess salt is carried over into the cuvette.

Personally, I prefer chemical transformation. Although it takes around half-an-hour longer, I find that the results are more predictable and I like the fact that a greater volume of DNA can be added if the concentration is too low. You can’t increase the volume of DNA with electroporation because of the risk of adding too much salt to the solution. Although the commonly cited drawback of chemically transformation is its lower efficiency, you can now buy ultra-competent cells, such as Stratagene’s XL10-gold, so this is no longer so much of a problem.

Articles in this series:

Originally published on September 18, 2007. Revised and updated on July 11, 2016.


What is DNA transformation

Plasmid or vector transformation is the process by which exogenous DNA is transferred into the host cell. Transformation usually implies uptake of DNA into bacterial, yeast or plant cells, while transfection is a term usually reserved for mammalian cells. Typically the method for transformation of a DNA construct into a host cell is chemical transformation, electroporation or particle bombardment. In chemical transformation, cell are made competent (able to take up exogenous DNA) by treatment with divalent cations such as calcium chloride, which make the bacterial cell wall more permeable to DNA. Heat shock is used to temporarily form pores in the cell membrane, allowing transfer of the exogenous DNA into the cell. In electroporation, a short electrical pulse is used to make the bacterial cell temporarily permeable. Particle bombardment, is typically used for the transformation of plant cells. Gold or tungsten particles are coated with the DNA construct and physically forced into the cell by gene gun.


What is the difference between natural and artificial competent cells?

Competent cells can either occur naturally or cells can artificially be made competent.

Natural cell competence:

Natural cell competence is genetically determined, that is to say, a bacterium is genetically predisposed to take up free genetic material (genetically competent) that exists within their environment. Research has shown that competent bacterial cells can take in DNA from a variety of sources however, some bacteria show specificity or a preference for DNA fragments that are overrepresented within their genome.

DNA taken up by a naturally competent cell does not always become incorporated into the cell’s genome. Often, a fragment of DNA will be used for nutritional purposes. For example, DNA provides a cell a much-needed source of deoxyribonucleotides for replication. The determination of how DNA is used within a cell depends usually on the needs of the cell. Other factors include existing DNA damage within the cell and recombination ability of incoming DNA.

While a cell might be considered naturally competent, the state of competence is not necessarily a constant state. In fact, natural regulation of competence and transformation is important for protecting a cell. Regulation often occurs through environmental and biochemical signals.

For example, Streptococcus pneumoniae is a naturally competent bacterium however, its competence is prompted by quorum sensing, or detecting and responding to cell population density through gene expression. For natural transformation to occur, there must be donor DNA present in the environment. One study determined that when conditions were met, donor DNA came from a “sub-fraction” of the S. pneumonia population, while the other portion was competent, taking up the DNA.

Artificial (induced) cell competence

Whether through electroporation or chemical methods such as calcium chloride, the process of making competent cells creates temporary pores in a cell’s membrane in order for DNA to pass through. Recall, competence is a cell’s ability to take up foreign DNA from its environment, while transformation is the actual process of DNA uptake. Therefore, in order for a scientist to transform a cell, she must first make that cell competent. This is done by changing the cell in such a way that enables DNA to easily travel through the cell membrane.

What exactly makes a cell competent?

Since natural competence is genetically determined, these cells will be equipped with special machinery to facilitate natural transformation, and often will have biochemical and environmental signals to regulate competency.

In a lab setting, usually with E. coli, artificial cell competence is made possible through a chemical process or through electroporation. Both of these methods alter the cell membrane, creating temporary pores that allow DNA to enter the cell.


Injection

A common way of introducing foreign DNA into plants is to physically inject the DNA with a gene gun. The concept of the gene gun is to coat microscopic particles of gold or tungsten with the foreign DNA. These particles are then loaded into the gun, which contains pressurized helium gas. A release of the gas propels the DNA-coated particles out of the gun like bullets. These particles penetrate the cell walls of plants and release the foreign DNA, which is now part of the plant cell. Gene guns can be used directly on the leaf of a plant or on plant cells that have been isolated from ground-up plant tissue.


Watch the video: Transformation Vs Transfection (June 2022).


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