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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

bacteria growth curve
sani Reply
No bacteria's don't grow curvey. Curve is a shape for a bacteria not growth
KINGSFORD
What is the difference between eukaryotes and prokaryotes?
KINGSFORD Reply
What's a multicellular organism?
KINGSFORD
multicellular organisms is an organism composed of many cells which are to varying degree integrated and independent.
Ruth
Boiling of milk to kill germs is called?
Kasigwa
pasteurization
Aiyedun
pasteurization
Katta
kadha
Katta
pasteurization
Gomathi
what is microscopic
monish
Pasteurization
Hosy
what are Flora
Hosy
you mean like the normal flora?
Tenucharity
pasteurization has to do with killing of the harmful microbes present for safe consumption. Eg. pasteurized yoghurt beside the bacteria that are supposed to be present, there may be harmful ones that have been killed
Tenucharity
Can someone explain flora
KINGSFORD
what is cell?
Anita Reply
the cell is the functional and structural unit of the body
Ruth
yes ur right
Kasigwa
who add me
Wali
hy
Katta
hy
Shivangi
basic structural, functional, and biological unit of all known living organisms. A cell is the smallest unit of life.
Haru
hi
Ruth
hi
Haru
coming Oct 2 I have a exam on microbiology
Haru
okay all the best
Ruth
hi
sachin
hi
Ruth
plz bro send question of microbiology
umesh
structural and functional unit of the life
monish
this is not the correct answer
Alanna Reply
why
Hayat
why not
Zaajid
what is the correct answer?
vannessa
where is question
Zaajid
ask you question
Zaajid
What are mitochondria
Hayat
what is mitochondria
Hayat
who is the father of microbiology
T-
not
Hayat
Antonie Van Luweenhoek
Ravikumar
Mitochondria is the powerhouse of the Cell. And is the cell organelle which helps cell in generation of energy by Cellular Respiration
Ravikumar
Mitochondria are threadlike structuresbounded by double membrane,and serves as the power house of the cell
Felicity
can I get notes on introduction to microbiology just started I have no idea please
rita
Mitochondria is a membrane bounded structure, it's sausage in shape and has inner and outer membrane. The inner membrane is folded at the surface called cristae which project to form matrix. They function as the power house for the cell.
KINGSFORD
Rita are you a medilab student?
KINGSFORD
the father of microbiology is Luis pasture
Able
Erythromycin--------(+++) Cefuroxine ------------(++++) Chloramphenicol --(++) Vancomycin ---------(++++) please what does the above result means. under urine culture and sensitivity?
Kwame
what is a vacual
Star Reply
What is microbiology
Nji Reply
it is the study of microscopic organisms and basically their relation to the environment
Tenucharity
what is elements
Erneet Reply
an Element is a substance that can not be further decomposed by ordinary chemical means.
vannessa
can i get notes of biochemistry
Sneha Reply
yep why not
Abdullah
what is microbiology?
Gayanjali Reply
what is malaria
Gayanjali
bios- life/living things micro- small things that cannot be seen with the naked eye In other words the study of living things that cannot be seen with the naked eye but with the help of a microscope
Coltuneac
logos- science
Coltuneac
it is 10 to the power minus 6 biology which studies life forms on those scales including metabolism , physiology, nutrition requirements
barun
what are prokarytes
NDIMUKIKA Reply
A prokaryotic is a unicellular organanism that lacks a membrane bound nucleus ,mitochondria or other any membrane bound organelle. .......
Maryam
in prokaryotic nucleus is not developed and cell organelles are absent
Harshad
how is one who is feeling blockage in the chest with mucus be diagnosed
Essy Reply
be talking sepitol every morning
Ikenna
is that all?
Ven.phumie
What is prions
Sangeeta Reply
Prions are microscopic organism which have no nucleic acid, no nuclear bounded membrane and affect the brain of animals like sheep's and human such a CJD and Kuru
KINGSFORD
is tuberculosis a viral infection?
hildar Reply
No. It is a Bacterial Infection.
Orooj
what are zn staining
Evelyn Reply
full meaning zeil neelson staining
kezia
By using zn stain we diagnose / examine acid fast bacilli .
Deepak
explain hw bacteria grows
Kanyago Reply
Hw does bacteria grow?
Kanyago
Binary fission
Sangeeta
three successive phase comes in bacteria growth i.e lag phase/adaptation phase ,log phase/exponential phase ,death phase when it is in fermentation process .
Deepak
Bacteria's don't grow by binary fission, they're grow through Biofilm formation, through colonies and transformation of clear broth medium to a turbid suspension
KINGSFORD

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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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