<< Chapter < Page Chapter >> Page >

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

Got questions? Get instant answers now!

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

true

Got questions? Get instant answers now!

Fill in the blank

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

transfection

Got questions? Get instant answers now!

Short answer

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

Got questions? Get instant answers now!

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

Got questions? Get instant answers now!

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

Got questions? Get instant answers now!

Questions & Answers

intracellular vesicles are found in
Akshay Reply
how do i report widal slide results
Isaac Reply
how food can be used as a substrate for the growth of microorganisms
Ashi Reply
what is the importance of understanding chemistry in the field of microbiology and food technology?
Charmaine Reply
What is different between eukaryotes and prokcaryotic
Abia Reply
main difference is that eukaryotic cells possess membrane bound organelles
Iqra
prokaryotes are primitive organisms that doesn't contain membrane bound nucleus or any orgenelle while in eukaryotes membrane bound orgenelles and nucleus is present
Yashi
what s anatomy
jane Reply
its mean body structure, function, and systems
gazi
study of internal structure of living things
Falere
anatomy is the study structure of the made human
isir
anotomy is the actual study of body internally and externally Which include how itis made. for what and what is need of this
Iqra
la science anatomie c'est la science qui nous aide à étudier l'homme
Baraka
okay
Baraka
Ancestor are they real
Rapheal Reply
yes of course
David
yes please
Clark
50 50, depending on the accuracy of the clan records.
Vincent
okay
Baraka
Why protist is not a kingdom of Linnaeus 'S taxonomy?
Neha Reply
I dont know
Jeewraj
Linnaeus used Aristotle's criteria for dividing living organisms into kingdoms. Plants are immobile and insensitive, while animals are mobile and sensitive. the creatures he saw through the microscope were mobile, so he attributed them to animals.
Lad
name the different types of media use in lab to detect the micro organisms
Shehzadi Reply
The different types of media used in the lab to detect the microorganisms is known as cell culture..
Tean
solid media or broth is used to grow and detect microorganisms
Pohor
what is microbiology
Baraka
the study of large living organisms
Pratibha
the study of organisms which are micro in range
Vency
what is autoclaving?
Yashi
process for sterilization
Vency
is a machine used on the process of sterilisation
Clark
what is difference between hot air oven and autoclave as they both are used for sterilization ?
Vency
autoclave basically do moist heat sterilization while hot air oven do sterilization by dry heat.....
Yashi
what is the microbe
HUSSAIN
which method out of these two is best?
Yashi
why human have microbe
HUSSAIN
Hi
Sadam
hi to all
Sadam
depends on what to sterilize
Vency
microbes
Md
what is knowledge
HUSSAIN
well idea
Daniel
what are the importance of microbiologe to a nurse
Enny Reply
it enable a nurse to know the weight and height of a patient
Abotu
it helps the nurse in way as to give first aid to patient on which basis doctor will take diagonos
Iqra
disagree with both of you
Akhtar
ok
Abotu
Your Own idea
Abotu
it helps a nurse to be able to counsel a patient /client
Abotu
cell culture
Tean
Types of micro organisms
Jennifer Reply
Who discovered plant and animal cell
Jennifer
Robert Hooke
Martha
Father of microbiology
Jennifer Reply
Antonie van Leeuwenhoek
Satarupa
Why are mitochondria and chloroplasts unable to multiply outside of a host cell?
Iqra Reply
iqra mitochondria arises from the division of existing mitochondria and they are fused together. they move around inside the cell with the interactions of the cytoskeleton that's why mitochondria unable to multiply outside the host cells chloroplasts has its own separate DNA from the plant cell.
Rana
short not on medical microbiology
Massah Reply
Hy
Iqra
hope you fine
Arif
love you
Arif
Assalamualaikum
Suhaib
arai yeh micro kai sth kis ko love hua
Umer
icrobiology is the scientific study of these microorganisms. Microorganisms are those organisms that are too small to see with the naked eye and include things like bacteria, fungi, and viruses
Suhaib
iqra iqbal
Arif
What is gram syaining?
Arif
to differentiate between gram positive and gram negative bacty
Umer
True
Arif
where you are from
Arif
i m frm sopore..kashmir
Umer
i m frm baramulla .. Kashmir
Umer
and u
Umer
umer
Arif
and i m from afghanistan lovely country
Arif
yes
Umer
thanks
Arif
medical microbiology means study of microorganisms which are beneficial or harmful for the body. mostly which are studied under microscope by structure and shape appearance. e.g bacteria, viruses, bacteriophage, fungus hyphae and some blood and other body fluids parasites e.g plasmodium etc.
Rana
arif bhai gram staining in which we are given different dyes to the bacterial cell wall and cytoplasm and nucleus.e.g giemsa stain
Rana
Gram staining uses crystal violet to stain cell walls iodine as a mordant and a fuchsin or safranin counterstain to mark all bacteria
Umer
Gram staining uses crystal violet to stain cell walls iodine as a mordant and a fuchsin or safranin counterstain to mark all bacteria
Umer
Gram staining uses crystal violet to stain cell walls iodine as a mordant and a fuchsin or safranin counterstain to mark all bacteria
Umer
Omar baba crystal violet iodine 70% alcohol fuchicine or safranine after every step washing must be done
Rana
hello
Cristy
*Fuchsin stain
Cristy
*Safranin
Cristy
thanks
Ganesh
difference between epidermophyton trichophyton and microsporum
Ankita Reply
plz answer the question
Iqra
epidermophyton type of fungi causes superficial and cutaneous mycoses trichophyton is also fungi type including parasitic varieties causes dermatophytosis microsporum is also type of fungi causes dermatophytosis.
Rana
ok
Iqra
Is this app perfect for preparation of exam?
Ankita
Ankita I totally disagree with your respected opinion. it's just minor helpful
Rana
disagree by me ?
Ankita

Get the best Microbiology course in your pocket!





Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
Google Play and the Google Play logo are trademarks of Google Inc.

Notification Switch

Would you like to follow the 'Microbiology' conversation and receive update notifications?

Ask