Cloning has a variety of different meanings and methods in the science world. Some clones exist naturally, such as asexual organisms or bacteria. However, in the biotechnology industry, cloning refers to material that needs external help in the cloning process. Those who work in this field create copies, or clones, as small as DNA fragments and cells to complete living organisms.
A huge strength of working withDNA is the ease of creating recombinant DNA constructs.Custom DNA sequences can be constructed as templates for subsequent production of customized proteins. One way to drive expression of a piece of DNA is to insert thatDNA sequence into a plasmid vector.There are several online tools, called plasmid editors, which help identify where and how to insert the DNA at the right location in the vector.Typically, an expression vector will contain the gene of interest (GOI), a bacterial origin of replication i.e. a promotor, an antibiotic selection cassette for the plasmid host and an antibiotic selection cassette for E. coli.
To identify and amplify aGOI, DNA oligos are utilized for polymerase chain reaction (PCR). Oligos are small, synthetic fragments of DNA that are designed to match the beginning and end of theGOI. Oligos are typically 15-25 base pairs long with a sequence that matches a specific restriction enzyme cut site, which corresponds to a cut site in the desired vector. One oligo will match the DNA in the 5’ to 3’ direction and the other will match in the 3’ to 5’ direction. During PCR, the oligos will amplify the GOI and the resulting pool of DNA will have restriction enzyme cut sites on each end. A plasmid editor is useful for identifying the right sequence for the oligo and for deciding which restriction enzyme sequences to add. When you have designed oligos, they can be synthesized in vitro utilizing a number of products we carry or ordered from companies,such as Addgene, who will build the oligos and mail them to you for use in your experiment.
Choosing the right plasmid vector is critical. Different plasmid vectors contain different restriction enzyme cut sites, offering versatility for oligo design. Expression vectors will contain a promoter for expression of the desired protein. Lastly, most vectors will contain one or more antibiotic resistance markers that can be used to select for cells that have successfully taken in the plasmid DNA after bacterial transformation.
DNA can be extracted and purified from various sample types, such as cells, blood, tissue or serum. PCR is performed using custom designed oligos to amplify the GOI. Upon verification and extraction of the amplified fragment, the DNA is digested with restriction enzymes that are added to the sequence via the oligos during amplification.
The plasmid vector is then cut with the same restriction enzymes. After digestion with restriction enzymes, the DNA and the plasmid vector will have asymmetric overhangs called“sticky ends”. These sticky ends have high affinity for other sticky ends cut with the same enzyme and are used to ligate the DNA into the plasmid vector.The cut plasmid vector and the cut GOI are ligated together by incubation in a solution containing ligase. The ligase will bind the matching sticky ends cut by the restriction enzymes, creating a circular plasmid, called a recombinant plasmid.
The recombinant plasmid is transformed into competent cells, such as E. coli, for propagation and storage. Transformation is facilitated by creating pores in the membranes of competent cells, either through electroporation or heat shock. Recombinant plasmid permeates the membrane, entering the cytoplasm of the competent cell. If the vector contains an antibiotic resistant marker, the cells are then incubated in media with said antibiotic, so only those cells which contain the vector will survive. In this way, one can select for only those cells that contain the newly created plasmid vector.
After transformation, cells containing the gene of interest may be stored safely long-term in glycerol at -80˚C or lysed to collect DNA containing the GOIfor various experimental purposes.
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To express a protein of interest within a target mammalian cell environment, custom DNA is transfected into target cells.Common transfection methods include lipofectamine, calcium phosphate transfection, and lentiviral transduction.
In lipofectamine transfection, the custom DNA plasmid is suspended in serum-free DMEM with lipofectamine. After incubation, the mixture is added to cells. Transfection by lipofectamine is fast and easy but has relatively lower expression when compared to other methods.
In calcium phosphate transfection, 0.25 M of CaCl2is mixed with a small amount (1-2μg) of the DNA construct. This mixture is added to a tube containing phosphates and HEPES buffered saline. After incubation, the mixture is added dropwise to cells. The cells are then left to incubate 4-6 hours before being washed in fresh phosphate buffered saline. Calcium phosphate transfection is a time-consuming process, and the calcium phosphate mixture is highly toxic. Even so, calcium phosphate transfection results in high rates of expression, so it is often utilized when there is little genetic material to work with.
Lastly, lentiviral transduction utilizes viral vectors for cellular transfection. The viral vector cannot live outside of lentiviral-infected host cells. Host cells are transfected via either of the two above methods: lipofectamine or calcium phosphate transfection. The host cell will then package the construct into lentiviral particles. Particles are collected and added at equal ratio to cells with a transduction agent.This process is time-consuming, and the use of viruses may require an upscaling in the amount of necessary biosafety, requiring additional PPE and revised disposal protocols. However, lentivirus transduction has high efficiency and may be used to infect many types of cells.
Transfection typically refers to the introduction of DNA into mammalian eukaryotic cells. However, DNA may also be placed into prokaryotic cells or other types of cells.Bacterial transformations can be facilitated through heat shock, electroporation, or through chemical means like lithium acetate treatment. Yeast and insect cells can also be used, transformation requires similar chemical methods or electroporation.
Once expressed in the target cells, proteins must be verified. Often, proteins are verified by western blot. Some fusion protein shave fluorescent markers that may identified by scanning. A specifically chosen phenotypic effect of the protein of interest can also be used to determine successful transfection.
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1. Developing a primary culture
Primary culture refers to the cells that are isolated directly from the tissue of interest and proliferated until they reach confluence, or occupy all the available substrate. One method of acquiring cells for primary culture entails sampling from the tissue directly. Cells taken in this manner must be disaggregated using enzymatic or mechanical means before they are placed on the substrate.
After the primary culture reaches confluence, the cells have to be subcultured by transferring them to fresh growth medium. At this stage the cell culture is no longer considered primary and becomes secondary culture, or cell line. Cell lines derived from primary cultures are finite, which means they have a limited number of cell divisions that is a genetically determined. The loss of ability to proliferate is called senescence.
Another way of acquiring cell culture is by using an established cell strain. These are continuous, or immortalized, cell lines that have been mutated and lost the ability to undergo senescence. These cells can continuously divide and are optimal cell lines for prolonged studies. The most common immortalized cell lines used for primary culture include HeLa cells and HEK 293 cells, among others
2. Developing Cell Lines
Regardless of the cell line type, it is necessary to propagate the cell line by continuously passaging the cells into fresh media, a process known as splitting the cells. To split cells, cell media is aspirated via a vacuum and the cells are washed in warmed Phosphate Buffered Saline (PBS) to remove residual media. If the cells are anchorage-dependent and rely on adherence to a surface, the cells can be treated with trypsin, a chemical that causes cells to temporarily lose their adherence. Trypsin, however, is toxic and should not be applied for long.
Trypsin toxicity is neutralized by adding growth media. For mammalian cells, this growth media is typically DMEM supplemented with Fetal Bovine Serum (FBS) and antibiotics. From this solution, the media and cells can be partitioned, or split, into new cell culture plates. A common practice is to always maintain at least one plate for further splitting and use the rest to split into additional plates that will be used for experiments.
While the above example is for anchorage-dependent mammalian cells, a similar principle applies to other types of cells including those in suspension and spore colony culture. In suspension culture, cells from a developed culture may be split by transfer into fresh growth media. In colony culture, spores or colonies may be taken from a developed plate and streaked across fresh media plates
Different cells require different growth conditions. While conditions may vary from cell type to cell type, most cells are grown in incubators to maintain optimum growth conditions. Incubators have the ability to maintain an optimum temperature (37oC for most cells) and precise O2 and CO2 levels. Additionally, cells will require a growth medium that is unique to each cell type. Media contains the nutrients, hormones, and buffers for cells to grow and should be continually changed. The right media and incubation conditions are critical for cell growth.
3. Maintaining Cell Lines
Successful cell culture depends on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and viruses. Nonsterile supplies, media, reagents, airborne particles, unclean incubators, and dirty work surfaces are all sources of biological contamination.
Aseptic technique is the best barrier to prevent contamination by invasive microorganisms and should always be maintained. Aseptic technique includes:
- Only opening cell culture in a sterile environment such as a cell culture hood with working airflow.
- Sterilizing the work area before and after each handling of the cells with proper cleaning reagents. 70% ethanol is the most common choice.
- Using gloves, lab coats, and other PPE as needed to prevent contamination of the samples and maintain personal safety.
- All media and solutions should be opened and/or mixed within a cell culture hood.
- Using only sterile glassware and disposable pipettes. Dispose of pipettes after each use.
- Opening media and other containers only when ready to use.
However, even with proper aseptic technique, contamination can happen. In these cases, the incubator and cell culture hood should be thoroughly cleaned to prevent other samples from being contaminated. If contaminated cells or media are critical to work, they may be treated with antibiotics to kill the contaminants - though care should be taken as antibiotics may cross-react with the cells of interest. In most cases, the contaminated cells and media should be thrown out and prepared anew.
Long-term storage of cells is a useful way to backup any experiments relying on a certain subculture or strain of cells. The most common method is Cryopreservation. Cryopreservation requires a surplus of cells be taken from cell culture and mixed with a protective agent, typically DMSO or glycerol, before being stored below –130°C.