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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

what is bacteria
Ibrahim Reply
a member of a large group of unicellular microorganisms which have cell walls but lack organelles and on organised nucleus and ,including some which can cause disease
Nadiya
what is a germ
Ibrahim
a microorganisms,speciallay one which causes disease .
Nadiya
bacteria are a type of biological cells. They are unicellular. Bacteria were among the first life forms appear on the earth. they have a number of shapes, typically a few micrometers in length. They are present in of its habitata on earth.
isha
What is Auto clave?
Lovely
Autoclave is a pressure chamber used to sterilize equipments
praveen
hi
Lovely
n
vijaya
hii
chandi
what is a Laminar air flow?
isha
Laminar airflow cabinet is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples or any particle sensitive materials
praveen
Praveen Deepi.... thnq....
isha
what bacteria causes malaria
Silas
female anaphilis mosquitos!
Nadiya
malaria isn`t caused by a bacteria rather by a digenetic parasite named Plasmodium.
Online
plasmodium sp
SRUTHI
its caused by a plasmodium germ....
Cotilda
Malariya cycle
Mohit
what caused the expansion of the wound
Blessing Reply
Robert Koch ka Pura naam kya h?
isha Reply
the theory of Koch,'s Postulation are: 1_Any disease must found microorganism 2_Microorganism can grow it outside pure culture 3_ Pure culture can inoculated to susceptible host and can cause typical disease. 4_ Microorganism can reisolated from inoculated disease from susceptible host.
Algur
ok
Kwame
that good
Ifeoma
please I want a detailed explanation to " G6PD result being Partial Defect" of a young lady.
Kwame
a.o.a can u please tell me five name of Gram +be bacteria
Tahira Reply
a.o.a...please send me five name of Gram +ve bacteria
Tahira
a.o.a...please send me five name of Gram +ve bacteria
Tahira
a.o.a...please send me five name of Gram +ve bacteria
Tahira
staphylococcus ,streptococcus,lactobacillus,mycobacterium,clostridium, bacillus,enterococcus.
Nadiya
thank u so much
Tahira
1. Staphylococci 2. Mycobacterium tuberculosis 3. Bacillus anthrasis 4. Chlostridium 5. Diplococci
praveen
gram -ve mn knsy hn
Tahira
pseudomonas
Nadiya
wo bhi 5 hon
Tahira
salmonella,proteus,shigella,neisseria
Nadiya
wo bhi 5 hon
Tahira
Nadiya ap kia krti hn?
Tahira
bsc microbiology final year
Nadiya
1. Neisseria gonohorreae 2. E.coli 3. Salmonella typhi 4. Klebsiella pneumonia 5. Shigella dysenteriae
praveen
1. Neisseria gonohorreae 2. E.coli 3. Salmonella typhi 4. Klebsiella pneumonia 5. Shigella dysenteriae
praveen
1. Neisseria gonohorreae 2. E.coli 3. Salmonella typhi 4. Klebsiella pneumonia 5. Shigella dysenteriae
praveen
MSc zoolgy. 1st semester
Tahira
B.Sc microbiology 1st year
praveen
good
Tahira
mn vu sy hoon and u?
Tahira
davv
Nadiya
Vijaya College,R.V.Road,Basavangudi Bangalore-560004
praveen
oh!! bht khub
Nadiya
hi
Nouman
how are u
Nouman
thank u friend
Nouman
please define classification of bacteria
Nouman
calssification of bacteria are of 3 types: ●.on the basis of shap:1.spirilla 2.bacilli 3.cocci ●in the basis of gram stain : 1.gram -ve 2.gram +ve ●on the basis of oxygen demand: 1.aerobic 2.anaerobic.
Nadiya
detail se please
Nouman
paresention hai iseley
Nouman
jo bacteria hote hai pehle to woh 3 tarha se unka classification kiya jata hai .pehle y ki shape kya hai ....lakin shape hum tab hi define kar payenge jab hum unhe pehchanenge ki bacteria ko kaise jane ki woh kaise hote hai or unka classification kaise hota hai
Nadiya
to hum gram staining karte hai
Nadiya
gram staining ek aaisi technique hoti hai jise hun 2 large group m devide karte hai
Nadiya
or differenciate karte hai bacteria ko
Nadiya
based on thir diff. cell wall constituents
Nadiya
gram staining ko 2 procedure mai distinguish between kiya gaya hai
Nadiya
gram positive and gram negative
Nadiya
phir
Nouman
gram +ve and gram -ve groups by colouring these cells red or violet colour.
Nadiya
red jo cells hogi woh pink bhi ho sakti h because stains ki wajah se uski quantity ki wajah se
Nadiya
gram positive bact.stain violet due to presence a thick layer of a peptidoglycon in theri cell walls are stained with.. or gram -ve bact.stain red .which is attribute to a thinner peptidoglycan wall.
Nadiya
ab hum unhe identify karenge microscope mai
Nadiya
then fir hum identify karte karte hum shape dekhenge
Nadiya
gram positive cocci or rod shaped k honge .or garm negative cocci or rods or spirochaetes honge .......lakin inke colour ki wajah se hum pehchanenge ki red or pink aaya to gram negative .or agar violet aaya to positive
Nadiya
ok?
Nadiya
fir
Nouman
or bhi types k hote hai bacteria jaise sphirical "cocci",rod "bacilli",spiral"spirilla",comma shape k jo honge woh"vibrio",or corkscrew shape k jo honge woh "spirochaetes.
Nadiya
fir
Nouman
or oxygen k basis pr dekhenge agar apan to woh :aerobic jo o2 ki presence m honge or anaerobic jo honge woh without oxygen ki presence mai servive karenge .sabhi micro organisms banate h energy cellular respiration k through. lakin aerobic and anaerobic k basis pr bhi diffrenciate hote hai yeh .
Nadiya
Streptococcus Staphylococcus Corynebacterium Clostridium
Srinivas
yeh 3no calssification batadiye detail mai ab aap net p or seach kar le
Nadiya
fir
Nouman
ok thank u so much
Nouman
کتنے پیارے اور چھوٹے سے خواب ہیں آپ کے ۔ الله پاک آپ کے سپنے سچ اور پورے کرے ۔ آمین ۔۔۔۔۔۔۔۔۔۔۔💝
Nouman
my pleasure!
Nadiya
acha ji ap ho kaha se
Nouman
Hyderabad
Srinivas
telangana
Srinivas
Can hiv virus survive in mosquitoes?
Mad
hiv virus cannot survive in mosquitoes.
Nadiya
Lol
Number
What,s the correct answer?
Nadiya
:-)
Nadiya
We will also know?what's the right answer !
Nadiya
Good going people
Number
Ok tell me all the conditions where a gram positive organism appears wrongly as gram negative organism in microscopy
Number
Staphylococcus Listria 2.Streptomyces 3. Streptococcus 4. Clostridium 5. Propionibacteriu
Basem
We know answer!
Lovely
ok
Basem
but you tell me first :can hiv virus survivr in mosquitoes? i dont know what's the correct answer !
Nadiya
No it can't.... Coz retroviruses require CDt4 receptors for replication which aren't found in mosquitoes
Number
Samjhi?
Number
yaaaa .......jazakallah !
Nadiya
well done doc.!
Nadiya
Good
Number
scale up fermentation ? define plz !
Nadiya
U can find all these answers easily on Web. Discuss something which is unique
Number
what is the gram negative
Xaviery Reply
what is enzyme immobilizatio ?
Nadiya Reply
the process of decreasing the rate of enzyme activity.
Edward
shukriya edward knevy .thnks .
Nadiya
sorry thi one is about enzyme inhibition
Edward
enzyme immobization is imprisonment of enzyme in a matrix.
Edward
i am confused
Nadiya
about what
Edward
ok .....i understood ur last one msg .
Nadiya
thanks .
Nadiya
Hallow
Chimo
my pleasure
Edward
can you helpe me please 🤗🤗
Chimo
?
Nadiya
but why
Nadiya
imuhhe patta nhi aapka ki aap kon ho to mai kaise no. send karu aapko or kyu karu ? wajaah to bataaa .
Nadiya
mujhe pata nhi
Nadiya
I want to learn microbiology
rakesh
this app is very benifit to learn microbiology .
Nadiya
Man Topics for Microbiology
rakesh
Please tell me
rakesh
first.u clear ur basic concept in microbiology .
Nadiya
Secondly what should you do
Nancy
u can start ur studies in book by dubey and maheshwari author.
Nadiya
what is primary and secondary screening ?
Nadiya Reply
which scope in the field of dmlt corse ? snd plz explain your experiences with me !
Nadiya
Country?
Number
india
Nadiya
Depends on place
Number
your birth place ?
Nadiya
whats your profession ?
Nadiya
scope of microbiolgy?
Nadiya
Clinical microbiologist
Number
M a doctor
Number
Dmlt isn't that good in india
Number
Bakwas hai
Number
but why ?
Nadiya
whats ur counter plz give m answer ?
Nadiya
Coz i know
Number
Saturation
Number
oh .i get it.
Nadiya
but what choose i ?in this field?
Nadiya
Hmm
Number
Wats ur basic qualification
Number
Good evening all
Ngozi
microbiology
Nadiya
evening
Dennis
good evening 2 u
Nadiya
What microbiology?
Number
bsc final year in micro.......
Nadiya
Do msc n PhD then.... Its better than dmlt n ll
Number
but i dont do this
Nadiya
what Cadillac
Topcy
number@......u r a doctor ?
Nadiya
really
Nadiya
plz explain the defination of scale up of fermentation ?
Nadiya
how do micro biology aids pharmacy
Dennis Reply
please check Google.
Nadiya
bcz very interested topic are able in goggle site
Nadiya
I'm Jeremiah what are the step that involved Gram staining
Jeremiah Reply
place slide with heat fixed smear on staining
Nadiya
smear with crystal violet and let stand for 1 min
Nadiya
and then after 1 min gently wash rinse with tap water...than smear the grams idone and stand for 1 min
Nadiya
again rinse with tap water.then,rhe smear will appear as a purple circle on the slide
Nadiya
then ,decolarize 95%with ethyl alcohol and acetone .till the slide slightly and apply tge alcohol deop by drop for 5 to 10 sec.
Nadiya
and then the alcohol until runs the almost clear .carefully not to over decolorization
Nadiya
immideatly rinse with water.
Nadiya
view the smear using a light microscope under oil immersion
Nadiya
and then complete 😊
Nadiya
.
Nadiya
what is the relation between aspergillus and vinegar.
Asad
can anyone send me the procedure of using autoclave.
Asad
yeh i will....but i can send a site on google !then u can read easily and understand it the procedure of autoclave ? ok .
Nadiya
***google autoclave procedure.
Nadiya
and u write in google site ......procedure of using an autoclave
Nadiya
can anyone send me the calculation and formula for spread plate technique cfu/ml
Ankita
@Nadiya... I think u have missed the last step of gram staining after decolonization with alcohol we have to counter stain it with either basic fucshin Or Saffranin.
Ankita
yaa ofcourse ...thank u so much ankitaa.
Nadiya
hi asghar
Nadiya
who are u ? and where are u live from ?
Nadiya
Ideally we should use 5 second technique while making one slide of Grams stain
Number
what is scale up of fermentation ?
Nadiya
the most commonly used microorganisms in alcohol fermentation is?
Shaikh Reply
Saccharomyces cerevisiae
Online
saccharomyses
Nadiya
Yeast is the most commonly used microorganism for ethanol productionby fermentation. ... Besides S. cerevisiae, other examples of yeastsused for ethanol production are Schizosaccharomyces pombe, Kluyveromyces lactis, Candida spp., Pichia spp that are able to ferment...
bazi
Ethanol fermentation using the hydrolysate obtained after the saccharification of biomass is the last step in lignocellulosic bioethanol production process. The hydrolysate contains large amount of fermentable sugars that can be directly used by the ethanologenic microorganisms. Yeast is the most co
Najeebul
saccharomyces
Nadiya
yeast
Nadiya
yeast
Veronica
A.o.a aspergillus aur vinegar ka kia relation he.?
Asad
Hmm
Number
what is difference between primary host and secondary host ?
Edward
what is primary screening and secondary screening ....hmm?
Nadiya
primary and secondry host ...best example of female anaPhilis .and disease -maleria !
Nadiya
i am asking about difference not example
Edward
Primary host- the host in which the adult parasite lives, and reproduces sexually. Secondary host- the host in which the immature forms of parasite lives
bazi
primary host -the host in which the adult parasite lives ,and reproduces sexually.and the second host -host in which the immature forms of parasite lives. okyyyy?
Nadiya
synthesis of DNA from a single strand of RNA
Veronica Reply
In this initially , a single stranded RNA is taken into a eppendorff tube as a template for synthesis of Double stranded DNA and dNTPs are added along with reverse transcriptase enzyme . All should be placed in buffered solution. Then above mixture is run on RTPCR machine.
Ravikumar
In which initially double stranded RNA- DNA hybrid is formed and later multiple copies of Double stranded DNA is Formed in number of cycles.
Ravikumar
exactly
Nadiya
what is a virus?
abdulwali Reply
What is DNA transcription
abdulwali
virus is not a bacteria ...it is present in our body fluid .the protein are not composed and work in a proper manner the virus present quikly .
Nadiya
i refer a book name .u find ur question answers .12 or 11th book
Nadiya
DNA trancription is a process the imfirmation are transfer into dna to dna and dna to rna is called dna transcription
Nadiya
in other words,dna transcription ,:in thich a particular segment of dna is copied into rnaand especially mRNA by the enzyme rna polymerase .
Nadiya
why most virus are of RNA genome?
Mika Reply
Good Question❓
MALESELA
its due to their fastidious nature.
Kwame
enlist types of bacteria on tha basis of arrangement of tha flagella
Mihir Reply
monotrichous:vibrio cholerae, amphitrichous,lophotrichous,and last peritrichous.
Nadiya
econom emportance of bacteria
Japhet Reply
We can get agar which is used in microbiological media
Kashif
cause disease
Mika
industrial use.
Nadiya

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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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