Cloning

Cloning is the process of producing cells with identical or virtually identical DNA, either naturally or artificially.

In nature, many organisms produce clones through asexual reproduction. Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or DNA fragments.

1. Planning recombinant DNA constructs

Unlike proteins, DNA is simple to construct synthetically or in vitro. Therefore, custom DNA sequences can be constructed as templates for subsequent expression of customized proteins. These custom DNA sequences are commonly expressed in plasmids,which are circular DNA constructs.

Plasmid editors are available online and may be used to view and edit the DNA of plasmids. To begin creating a new DNA construct, two samples are required: the DNA of the plasmid being used and the DNA of the protein to be expressed or changed. The plasmid editor is then used to plan the insertion of the protein of interest into the plasmid by designing DNA oligos.

DNA oligos are small fragments of DNA that will be synthetically designed to match the DNA sequences of both ends of the protein of interest. The components comprising the oligo are:

• The sequence matching the last 15-20 nucleic acids of one end of the protein of interest.
• A sequence matching a restriction enzyme cut site.Take care the same site is not within the DNA of the protein of interest.
• Filler DNA to allow a better anchor for the digestion enzyme to cut.

These DNA oligos will flank the protein of interest and will be used to amplify out the DNA of the protein. The cut sites will be matched up with respective cut sites on the plasmid. In a plasmid editor, this can be seen as the plasmid sequence being interrupted by the insertion of the protein’s DNA sequence in-between two restriction enzyme cut sites. DNA oligos can be synthesized in vitro.

Choosing a plasmid for the protein of interest to be inserted into is critical. For most, acquiring a cloning plasmid will be as simple as ordering one from a company. Ideal plasmid vectors contain many restriction enzyme cut sites for versatility in choosing which cut sites to insert into the oligos. Additionally, expression vectors will contain a promoter for expression of the desired protein. Care should be taken when designing the oligos to orient the cut sites such that the protein is oriented in a 5’-3’ orientation relative to the promoter. Lastly, most expression vectorswill contain an antibiotic resistance marker that can be used to select for cells that have successfully taken in the plasmid DNA.

2. Creating custom DNA for insertion

confirmation

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DNA can be extracted and purified from the cell containing the DNA of the protein of interest. PCR can then be performed using the designed oligos to amplify out the proteins of interest from the rest of the DNA (See PCR for more details). Upon verification and extraction of the amplified fragment, the DNAwill be digested with the restriction enzymes matching the cut sites of the designed oligosin a 37oC water bath.

The plasmid vector must also be cut with these same restriction enzymes. The restriction enzymes cut the DNA at the specific cut site in such a way that forms asymmetrically cut “sticky ends”. These sticky ends have high affinity for other sticky ends cut with the same enzyme and are used to ligate the protein DNA into the plasmid vector.

Once cloning is complete, the plasmid is inserted into competent cells, such as E. coli,for propagation and storage in a process known as transformation. Transformation is done by introducing pores into competent cells either through electroporation or throughheat shock.These pores allow the recombinant plasmid to permeate through the cell’s outer defenses into the cytoplasm.Thetransformedcells are then incubated in media containing the antibiotic that the plasmid is resistant to select for cells that have successfully taken inthe plasmid DNA.

After transformation, cells containing the gene of interest may be stored safely for the long-term in glycerol or lysed to collect DNA containing the gene of interest. DNA isolation is commonly collected through pre-made kits containing buffers and columns for DNA collection.

Cloning constructs

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3. Transfection of Custom DNA into cells

Tofinally express the protein of interest within their target cell environment, the custom DNA must be transfectedinto the target cells.Some common transfection methods for mammalian cells include lipofectamine, calcium phosphate transfection, and lentiviral transduction.

Lipofectamine transfection involves the mixing of the DNA construct suspended in serum-free DMEM and lipofectamine 2000 in serum-free DMEM. After incubating for 20 minutes, the combined mixture is then added to the cells. Transfection by lipofectamine is fast and easy but has relatively lower expressionwhen compared toother methods.

In calcium phosphate transfection, .25 M of CaCl2is mixed with a small amount (1-2μg) of the DNA construct. This is added, dropwise, to a tube containing2mM phosphates in 2x HBS.

After 15 minutes of incubation, this combined mixture is added dropwise to cells. The cells are then left to incubate 4-6 hours before being washed in fresh PBS and having their media changed. Calcium phosphate transfection can be a time-consuming process.Additionally, the calcium phosphate mixture is highly toxic if applied too quickly and can kill the cells. Nevertheless, calcium phosphate transfectionhas a high expression rate and may be used when there is not a large amount of the DNA construct.

Lastly, lentiviral transduction utilizes viral vectors to transfect target cells. As viruses cannot live outside host cells, lentiviral-infected host cells are needed to produce the virus. These cells can be transfected with either of the two previously discussed transfection methods (lipofectamine or calcium phosphate) to insert the DNA construct. The host cell will then package the construct into lentiviral particles. These particles can be collected and added at an equal ratio of virus to cells along with a transduction agent (such as 15µg/mL Polybrene) to target cells the lentivirus will infect and express the protein of interest. This process is time-consuming. Additionally, the use of viruses may require an upscaling in the amount of biosafety that is needed and may thus require additional PPE and revised disposal protocols. However, lentivirus transduction has high efficiency and may be used to infect any cell infectable by lentivirus.

Transfection typically refers to the introduction of DNA into eukaryotic cells. However, DNA may also be placed into other types of cells as well. Transformation into non-animal cells may be done with the same DNA. Bacterial transformations, like the one described in “Creating custom DNA for insertion”, may be done through heat shock by applying extreme pressure differences to shock proteins into accepting foreign DNA. They may also be done through electroporation or through chemical meanssuch as lithium acetate. Insect and yeasttransformations may also be doneusing similar electroporation and chemical methods.

Once expressed in the target cells, proteins must be verified. Typically, proteins would be verified by western blot. However, some fusion proteins may have fluorescent markers that may identified by scanning. If the protein has a phenotypic effect it may be utilized to distinguish successfully transfected cells as well.

** The information displayed is only for general understanding only and cannot be used as a sole source for any research purposes.

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