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Electroporation

Compared to bacterial cells, eukaryotic cells tend to be less amenable as hosts for recombinant DNA molecules. Because eukaryotes are typically neither competent to take up foreign DNA nor able to maintain plasmids, transfection of eukaryotic hosts is far more challenging and requires more intrusive techniques for success. One method used for transfecting cells in cell culture is called electroporation . A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a short-lived positive charge on one side of the cell’s interior and a negative charge on the opposite side; the charge difference draws negatively charged DNA molecules into the cell ( [link] ).

A diagram showing electroporation. The first panel reads: introduce the gene into the cell. A cell with a distinct plasma membrane is shown and recombinant DNA is on the outside. The next panel reads: apply the electric pulse; pores form in the cell membrane and the gene enters. The image shows holes in the plasma membrane. Positive charges are inside the holes and negative charges are on the outside. Recombinant DNA pieces move into the cell. The final panel reads: after the electric pulse, the pores reseal and the gene remains in the cell. The diagram shows a continuous plasma membrane again and recombinant DNA both inside and outside the cell. The recombinant DNA inside the cell is labeled “introduced gene”
Electroporation is one laboratory technique used to introduce DNA into eukaryotic cells.

Microinjection

An alternative method of transfection is called microinjection . Because eukaryotic cells are typically larger than those of prokaryotes, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette, as shown in [link] .

A micrograph of a microinjection needle poking through the plasma membrane of a cell and into the nucleus.
Microinjection is another technique for introducing DNA into eukaryotic cells. A microinjection needle containing recombinant DNA is able to penetrate both the cell membrane and nuclear envelope.

Gene guns

Transfecting plant cells can be even more difficult than animal cells because of their thick cell walls. One approach involves treating plant cells with enzymes to remove their cell walls, producing protoplasts. Then, a gene gun is used to shoot gold or tungsten particles coated with recombinant DNA molecules into the plant protoplasts at high speeds. Recipient protoplast cells can then recover and be used to generate new transgenic plants ( [link] ).

a) a diagram of a gene gun. The gun barrel points towards a plant protoplast. A pulse of helium pushes microprojections (gold or tungsten particles coated with recombinant DNA molecules) through the barrel and into the plant cell. b) a photograph of a gene gun; it is the size and shape of a hair-dryer but with a much narrower opening.
Heavy-metal particles coated with recombinant DNA are shot into plant protoplasts using a gene gun. The resulting transformed cells are allowed to recover and can be used to generate recombinant plants. (a) A schematic of a gene gun. (b) A photograph of a gene gun. (credit a, b: modification of work by JA O'Brien, SC Lummis)

Shuttle vectors

Another method of transfecting plants involves shuttle vectors , plasmids that can move between bacterial and eukaryotic cells. The tumor-inducing (T i ) plasmids originating from the bacterium Agrobacterium tumefaciens are commonly used as shuttle vectors for incorporating genes into plants ( [link] ). In nature, the T i plasmids of A. tumefaciens cause plants to develop tumors when they are transferred from bacterial cells to plant cells. Researchers have been able to manipulate these naturally occurring plasmids to remove their tumor-causing genes and insert desirable DNA fragments. The resulting recombinant T i plasmids can be transferred into the plant genome through the natural transfer of T i plasmids from the bacterium to the plant host. Once inside the plant host cell, the gene of interest recombines into the plant cell’s genome.

A diagram of transformation of a plant cell using the Ti plasmid. A micrograph of rod shaped cells labeled Agrobacterium tumefaciens. Plasmids are isolated from these cells. The plasmid (Ti plasmid) has a region labeled T-DNA region. A gene of interest from the cellular DNA is inseted into the T-DNA region. The transformed recombinant DNA is the returned back to A. tumefaciens. The bacterium then infects the plant cell. This inserts the gene of interest and resuts in a recombinant plant.
The T i plasmid of Agrobacterium tumefaciens is a useful shuttle vector for the uptake of genes of interest into plant cells. The gene of interest is cloned into the T i plasmid, which is then introduced into plant cells. The gene of interest then recombines into the plant cell’s genome, allowing for the production of transgenic plants.

Viral vectors

Viral vectors can also be used to transfect eukaryotic cells. In fact, this method is often used in gene therapy (see Gene Therapy ) to introduce healthy genes into human patients suffering from diseases that result from genetic mutations. Viral genes can be deleted and replaced with the gene to be delivered to the patient; William S.M. Wold and Karoly Toth. “Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy.” Current Gene Therapy 13 no. 6 (2013): 421. the virus then infects the host cell and delivers the foreign DNA into the genome of the targeted cell. Adenoviruses are often used for this purpose because they can be grown to high titer and can infect both nondividing and dividing host cells. However, use of viral vectors for gene therapy can pose some risks for patients, as discussed in Gene Therapy .

  • What are the methods used to introduce recombinant DNA vectors into animal cells?
  • Compare and contrast shuttle vectors and viral vectors.

Key concepts and summary

  • Biotechology is the science of utilizing living systems to benefit humankind. In recent years, the ability to directly alter an organism’s genome through genetic engineering has been made possible due to advances in recombinant DNA technology, which allows researchers to create recombinant DNA molecules with new combinations of genetic material.
  • Molecular cloning involves methods used to construct recombinant DNA and facilitate their replication in host organisms. These methods include the use of restriction enzymes (to cut both foreign DNA and plasmid vectors) , ligation (to paste fragments of DNA together), and the introduction of recombinant DNA into a host organism (often bacteria).
  • Blue-white screening allows selection of bacterial transformants that contain recombinant plasmids using the phenotype of a reporter gene that is disabled by insertion of the DNA fragment.
  • Genomic libraries can be made by cloning genomic fragments from one organism into plasmid vectors or into bacteriophage.
  • cDNA libraries can be generated to represent the mRNA molecules expressed in a cell at a given point.
  • Transfection of eukaryotic hosts can be achieved through various methods using electroporation , gene guns , microinjection , shuttle vectors , and viral vectors .

True/false

Recombination is a process not usually observed in nature.

false

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It is generally easier to introduce recombinant DNA into prokaryotic cells than into eukaryotic cells.

true

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Fill in the blank

The process of introducing DNA molecules into eukaryotic cells is called ________.

transfection

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Short answer

Name three elements incorporated into a plasmid vector for efficient cloning.

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When would a scientist want to generate a cDNA library instead of a genomic library?

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What is one advantage of generating a genomic library using phages instead of plasmids?

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Questions & Answers

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Toxicity
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they are eukaryotic and most lived in fresh water. they are photosynthetic that's, they contain chlorophyll and store starch in discrete chloroplast.
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e.coli
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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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