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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

Explain about enzyme transportation
Shahla Reply
Enzyme transportation
Shahla
it looks reallllyyyyy coooooooollll. i love enzymes theyre so cooll and i also like protein transportation so i think it would be really cool so an enzyme transport something so ill do some research and come back to ya in that
maxo
they be ridin' dirty
Bethany
what is the infectious disease process
Patience Reply
what are differences between endotoxins and exotoxins
sabote Reply
endo toxins work in the nuceus. i think
maxo
tell me if im right tho
maxo
Exotoxins are toxic substances secreted by bacteria and released outside the cell. Both gram positive and gram negative bacteria can produce and secrete exotoxins. Whereas Endotoxins are bacterial toxins consisting of lipids that are located within a cell. Only lysed gram negatives.
Abdi
Remebr the Lipid A portion of LPS is what's toxic.
Abdi
oh yeah. thanks
maxo
Your welcome :)
Abdi
How did you learn this?
maxo
For me personally the best book is 'microbiology made ridiculously simple'
Abdi
I got my basics from there and slowly added information from other sources.
Abdi
thats cool! yeah i like microbiology too! especially the molecular proteins theyre sooooooooooo cool!
maxo
what are the prokaryotic
Lungu Reply
prokaraytotic is a unicellular organizm that lacks membrane bound nucleus
Zaajid
and whats eukaryotic
abdiqani
eukaryotic cell are cell which contain anuclues and organells
Zaajid
eukaryotes are the cells that have organells which are protected by membranes
maxo
eukaryotic is are multicellular organisms which are open nucleus.
Serah
Explain on the Francisco reddi did to prove the theory of spontaneous generation
Diana Reply
what is parasite
Abdirizack Reply
parasite are organisms feeds on a host for food and survival.e.g round worm for animal, Dodder for plant parasites.
Serah
parasite are organisms that feeds on a host for food and survival.e.g round worm for animal and mistletoe for plant parasites.
Serah
parasite are organisms that feed on their host
Cylla
designing of aseptic area
Aashish Reply
I don't know
Abdirizack
what is rickettsia
DENNIS Reply
what is microbiology
Erasto
Is the science that works with microorganisms.
Rose
richettsa is small microorganisms that cause disease in human like typhus; they are like viruses that can grow only inside living cells, they're transmitted by mites, ticks or lices.
Rose
what is plasmid?
mavis
plasmid is extra chromosomal body present in bacteria...which have additional genetic functions example... antibiotic resistance genes....etc etc
Chaitanya
state the theory of spontaneous generation of micro oranisms and germ theory of disease
UKAMAKA Reply
what are the advantages of high note numerical aperture
Genius Reply
list if non flagellated pritozoa
Mepung Reply
Can someone that's understanding of the Kreb Cycle please explain & breakdown it down to me in the simplest way without giving me the dictionary version or Google version. Basically in there own words of knowledge....!
Kisha
ok
lucas
please can someone help explain positive and negative feedback in simple term
Gum
negative feedback is the arresting of reaction or reverse of the reaction according to the response and postive feed back is the direct response without reversing or arresting a reaction.
Greet
OK
umar
pls can someone explained Kinney stone in memorising in shot time
umar
what tyoebif microorganism will be killed by antibiotic trwatmeant
Mary Reply
I don't really don't understand aerobic respiration anaerobic respiration and fermentation. Can someone explain & breakdown this in the simplest way please.
Kisha
Anaerobic Respiration in which foodstuffs are partially oxidized, with the release of chemical energy, in a process not involving atmospheric oxygen, such as alcoholic fermentation, in which one of the end products is ethanol.
Muhammad
aerobic respiration A type of respiration in which foodstuffs are completely oxidized to carbon dioxide and water, with the release of chemical energy, in a process requiring atmospheric oxygen.
Muhammad
please can someone explain what pseudomonas species and biofilm is?
uju
Fermentation is the growth of cells or microorganisms in bioreactors (fermenters) to synthesize special products. Fermentation in biochemistry refers to the biodegradation of carbon compounds by cells or organisms under anaerobic (lack of oxygen) conditions.
Muhammad
Please under which conditions does pathogens become established in the human tissues and can cause diseases
Atambilla
please the life cycle of plasmodium parasite
mavis
who is John Needham
Mary Reply
John Needham is one of the researcher in microbiology. He also experimented when scientists did not believe animals could arise spontaneously ,but did believe microbes could.
Gum
okay is he late
Mary
by the way what are the list of courses offered by a newly admitted student for microbiology
Mary
Needham's experiments with beef gravy and infusions of plants material reinforced this idea.
Gum
what drugs are given to a person with Otis nerve problem(ear problem)
Gum Reply
good morning to you all.
Muhammad Reply
Dr.A.K.S.P.G. college Akbarpur Ambedkar Nagar
Rajan
Shailesh Vishwakarma BSc 1st year
Rajan

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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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