GENE CLONING

ELECTROPORATION
TRANSFORMATION
SUBMITTED TO:
MAM DR. FARZANA SHAHIN

SUBMITTED BY:
AHMAD KAZMI (47) - GENE CLONING
M.YASIR (32) - GENE CLONING
QADEER KHAN (34) - ELECTROPORATION
YASIR IQBAL (02) - TRANSFORMATION
Gene Cloning:

• Techniques for gene cloning enable scientists to prepare multiple identical copies of
gene-sized pieces of DNA.
• Most methods for cloning pieces of DNA share certain general features.
• For example, a foreign gene is inserted into a bacterial plasmid and this recombinant
DNA molecule is returned to a bacterial cell.
• Every time this cell reproduces, the recombinant plasmid is replicated as well and
passed on to its descendents.
• Under suitable conditions, the bacterial clone will make the protein encoded by the
foreign gene.
• One basic cloning technique begins with the insertion of a foreign gene into a bacterial
plasmid.
GENE CLONING WITH DIFFERENT FACTORS:

• One goal may be to produce a

protein product for use.
• A second goal may be to prepare
many copies of the gene itself.
• This may enable scientists to
determine the gene’s nucleotide
sequence or provide an organism
with a new metabolic capability by
transferring a gene from another
organism.
Restriction Enzymes:

• In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or
other bacteria.
• Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences
and cutting at specific point in these sequences.
• Each restriction enzyme cleaves a specific sequence of bases called a restriction site.
• These are often a symmetrical series of four to eight bases on both strands running in
opposite directions.
• If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-
GAATTC-3
• Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a
staggered way creating single-stranded ends, sticky ends.
• These extensions will form hydrogen-bonded base pairs with complementary single-
stranded stretches on other DNA molecules cut with the same restriction enzyme
Recombinant DNA vectors:
• Recombinant plasmids are produced by splicing restriction
fragments from foreign DNA into plasmids.
• A plasmid is a circular piece of DNA found in bacteria and
contain genes.
• Plasmids can be used to insert DNA from another organism into
a bacterial cell.
• Then, as a bacterium carrying a recombinant plasmid
reproduces, the plasmid replicates within it.
• Bacteria are most commonly used as host cells for gene cloning
because DNA can be easily isolated and reintroduced into their
cells.
• Bacteria cultures also grow quickly, rapidly replicating the
foreign genes.
• Bacteria will also produce large amounts of the protein of
interest.
The process of cloning a gene in a bacterial plasmid can be divided
into five steps.

1. Isolation of vector and gene-source DNA:

• The source DNA may come from human tissue cells.

• The source of the plasmid is typically E. coli.
• This plasmid carries useful genes, such as ampR , conferring resistance to the
antibiotic ampicillin.

2. Insertion of DNA into the vector:

• By digesting both the plasmid and human DNA with the same restriction
enzyme we can create thousands of human DNA fragments, one fragment with
the gene that we want, and with compatible sticky ends on bacterial plasmids.
• After mixing, the human fragments and cut plasmids form complementary
pairs that are then joined by DNA ligase.
• This creates a mixture of recombinant DNA molecules.
3. Introduction of the cloning vector into cells:

• Bacterial cells take up the recombinant plasmids by transformation.

• This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant
plasmid DNA, other bacteria that have taken up other DNA, both recombinant and
nonrecombinant.

4. Cloning of cells (and foreign genes):

• We can plate out the transformed bacteria on a solid nutrient medium containing
ampicillin.
• Only bacteria that have the ampicillin-resistance plasmid will grow. 5. Identifying cell
clones with the right gene.
• In the final step, we will sort through the thousands of bacterial colonies with foreign
DNA to find those containing our gene of interest.
• One technique to test the clones of interest is to use DNA or Nucleic acid hybridization.
• This technique depends on base pairing between our gene and a short piece of DNA or
RNA with a complementary sequence to the gene called a Probe,
• The sequence of our RNA or DNA probe depends on knowledge of at least part of the
sequence of our gene.
• A radioactive or fluorescent tag labels the probe so that if it bind with our gene we can
detect it’s presence.
Reproductive cloning:

• Dolly was created by reproductive cloning technology, in a process called “somatic cell
nuclear transfer” (SCNT).

• Scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose
nucleus, and thus its genetic material, has been removed. The reconstructed egg
containing the DNA from of a donor cell must be treated with chemicals or electric
current in order to stimulate cell division.

• Once the clone embryo reaches a suitable stage, it is transferred to the uterus of a female
host where it continues to develop until birth.
DOLLY GENE CLONING:
Therapeutic cloning:

• Therapeutic cloning, also called “embryo

cloning”, is the production of human embryos
for use in research.
• The goal of this process is not to create clone
human beings, but rather to harvest stem cells
that can be used to study human development
and to treat disease.
• Stem cells are important to biomedical
researchers because they can be used to generate
virtually any type of specialized cell in the
human body.
 How can cloning technologies be used?
• Gene therapy can be used to treat certain genetic
conditions by introducing virus vectors that carry
corrected copies of faulty genes into the cells of a
host organism.

• Genes from different organisms that improve

taste and nutritional value or provide resistance
to particular types of disease can be used to
genetically engineered food crops.

• Reproductive cloning also could be used to

repopulate endangered animals or animals that
are difficult to breed.
What animals have been cloned?
• Scientists have been cloning animals for many years. In 1952, the first animal, a tadpole, was cloned.
• Since Dolly, researchers have cloned a number of large and small animals including sheep, goats,
cows, mice, pigs, cats, rabbits and a gaur.

Can organs be cloned for use in transplants?

• Scientists hope that one day therapeutic cloning can be used to generate tissues and
organs for transplants.

• The stem cells would be used to generate an organ or tissue that is a genetic match to
the recipient.

• In theory, the cloned organ could then be transplanted into the patient without the risk
of tissue rejection.
What are the risks of cloning?
• Reproductive cloning is expensive and highly inefficient.
• More than 90% of cloning attempts fail to produce viable
offspring.
• More than 100 nuclear transfer procedures could be
required to produce one viable clone.
• In addition to low success rates, cloned animals tend to
have more compromised immune function and higher
rates of infection, tumor growth and other disorders.
 Electroporation or electropermeabilization:
• Electroporation, or electropermeabilization, is a microbiology technique in which an electrical
field is applied to cells in order to increase the permeability of the cell membrane, allowing
chemicals, drugs, or DNA to be introduced into the cell (also called electrotransfer ).
• In microbiology, the process of electroporation is often used to transform  bacteria , yeast ,
or plant  protoplasts  by introducing new coding DNA.
• If bacteria and plasmids  are mixed together, the plasmids can be transferred into the bacteria
after electroporation, though depending on what is being transferred cell-penetratig peptides
 or CellSqueeze  could also be used.
• Electroporation is also highly efficient for the introduction of foreign genes into tissue culture
cells, especially mammalian cells. For example, it is used in the process of
producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy.
• The process of introducing foreign DNA into eukaryotic cells is known as transfection.
Electroporation is highly effective for transfecting cells in suspension using electroporation
cuvettes.
HISTORY:
In the 1960s, it was known that by applying an external electric field, a large membrane
potential at the two pole of a cell can be created.
In the 1970s, it was discovered that when a membrane potential reached a critical level, the
membrane will breakdown and that it could recover. 
By the 1980s, this opening was being used to introduce various of materials/molecules into
the cells.

IN VITRO AND ANIMAL STUDIES:

In vivo gene electrotransfer was first described in 1991 and today there are many preclinical
studies of gene electrotransfer. The method is used to deliver large variety of therapeutic
genes for potential treatment of several diseases, such as disorders in immune system,
tumors, metabolic disorders, monogenetic diseases, cardiovascular diseases, analgesia.
With regards to irreversible electroporation, the first successful treatment of malignant
cutaneous tumors implanted in mice was completed in 2007 by a group of scientists who
achieved complete tumor ablation in 12 out of 13 mice.
What cell type is appropriate for electroporation?

• ELECTROPORATION INTO MUSCLE OR

SKIN.
• Electroporation has been used successfully to
deliver plasmid DNA to a variety
of tissues in vivo (Heller et al., 2006a).
• Because of its physical nature, EP can be
applied to practically any cell or tissue.
Plasmid DNA in the appropriate diluent is
injected into the tissue.
Medical applications:

• The first medical application of electroporation was used for introducing poorly permeant
anticancer drugs into tumor nodules.Soon also gene electrotransfer became of special
interest because of its low cost, easiness of realization and safety. Namely, viral vectors
can have serious limitations in terms of immunogenicity and pathogenicity when used for
DNA transfer.
• A higher voltage of electroporation was found in pigs to irreversibly destroy target cells
within a narrow range while leaving neighboring cells unaffected, and thus represents a
promising new treatment for cancer, heart disease and other disease states that require
removal of tissue.
• Irreversible electroporation (IRE) has since proven effective in treating human cancer,
with surgeons at Johns Hopkins and other institutions now using the technology to
treat pancreatic cancer previously thought to be unresectable.
N-TIRE:
• A recent technique called non-thermal irreversible electroporation (N-TIRE) has proven
successful in treating many different types of tumors and other unwanted tissue.
• This procedure is done using small electrodes (about 1mm in diameter), placed either
inside or surrounding the target tissue to apply short, repetitive bursts of electricity at a
predetermined voltage and frequency.
• These bursts of electricity increase the resting transmembrane potential (TMP), so that
nanopores form in the plasma membrane.
• When the electricity applied to the tissue is above the electric field threshold of the target
tissue, the cells become permanently permeable from the formation of nanopores.
• As a result, the cells are unable to repair the damage and die due to a loss of homeostasis. 
• N-TIRE is unique to other tumor ablation techniques in that it does not create thermal
damage to the tissue around it.
Reversible electroporation:

• Contrastingly, reversible electroporation occurs when the electricity applied with the
electrodes is below the electric field threshold of the target tissue.
• Because the electricity applied is below the cells' threshold, it allows the cells to
repair their phospholipid bilayer and continue on with their normal cell functions.
• Reversible electroporation is typically done with treatments that involve getting a
drug or gene (or other molecule that is not normally permeable to the cell membrane)
into the cell.
• Not all tissue has the same electric field threshold; therefore careful calculations need
to be made prior to a treatment to ensure safety and efficac
Reversible electroporation:
• Contrastingly, reversible electroporation occurs when the electricity applied with
the electrodes is below the electric field threshold of the target tissue.
• Because the electricity applied is below the cells' threshold, it allows the cells to
repair their phospholipid bilayer and continue on with their normal cell functions.
Reversible electroporation is typically done with treatments that involve getting a
drug or gene (or other molecule that is not normally permeable to the cell
membrane) into the cell.
• Not all tissue has the same electric field threshold; therefore careful calculations
need to be made prior to a treatment to ensure safety and efficacy.
• One major advantage of using N-TIRE is that, when done correctly according to
careful calculations, it only affects the target tissue.
• Proteins, the extracellular matrix, and critical structures such as blood vessels and
nerves are all unaffected and left healthy by this treatment.
H-FIRE:

• A more recent technique has been developed called high-frequency irreversible

electroporation (H-FIRE).
• This technique uses electrodes to apply bipolar bursts of electricity at a high frequency, as
opposed to unipolar bursts of electricity at a low frequency.
• This type of procedure has the same tumor ablation success as N-TIRE.
• However, it has one distinct advantage, H-FIRE does not cause muscle contraction in the
patient and therefore there is no need for a paralytic agent. 
• Furthermore, H-FIRE has been demonstrated to produce more predicable ablations due to
the lesser difference in the electrical properties of tissues at higher frequencies.
Advantage of Electroporation:

• The advantage of elecotoroporation is its

applicability for transient and stable transfestion of
all cell type frthermore, because electoroporation
is easy and rapid .
• it is able to transfact a large number of cells in a
short time once opimum electoroporation
condition are determined.
Transformation:

Introduction:

 Genetic alteration of a cell by the incorporation of exogenous genetic material (exogenous

DNA).

 Transformation occurs naturally in some species of bacteria.

 It can also be done by artificial means in other cell.

 For transmission we use competent bacteria.

Transformation History:

• 1928 – Frederick Griffith transforms non pathogenic pneumococcus bacteria into a

virulent variety by mixing them with heat killed pathogenic bacteria.

• Transformation principle was demonstrated in 1944 by Oswald Avery ,Colin MacLeod

,and Maclyn McCarty, who showed gene transfer in streptococcus pneumonic was pure
DNA.

• Avery ,Macleod and McCarty call the uptake and incorporation of DNA by bacteria
transformation .
Competent:
cells that can be used for transformation are called competent. Types of
competent cells for transformation.

Types of competent cell:

• Chemically competent cells.

• Calcium chloride - treated = attachment of the plasmid.

• heat - shocked in a water bath =opens the pores of the cell membrane.

• entry of plasmid DNA from the buffer.

2nd type:

B:Electrocompetent cells .
• Using electroporation
• create pores
• genetic material enters the cells

Types of Transformation:
There are two types of Transformation.

• Natural transformation.
•
• Artificial transformation.
Natural Transformation:

In this case DNA take - up occur without outside help.

● Naturally competent bacterium - They can take up DNA from

the environment without requiring special treatment.

● About 40 species have been found to be naturally competent or

transformable.

● Examples : Bacilus subtilis, Streptococcus

pneumonia,Haemophilus influenza,Neisseria gonorrhoeae and
some species of marine cyanobacteria.
Artificial transformation:
Such transformation do by laboratory.

● Artificial competence is not encoded in the cell genes .

●It is induced by laboratory procedures in which cells are

passively made permeable to DNA ,using conditions that
do not normally occur in nature .

● These procedures are comparatively easy and simple and

are widely used to genetically engineer bacteria.

● Artificially competent cells of standard bacteria strains

may also be purchased frozen ready to us .
Basic procedure of transformation
(Artificial Transformation):

First:
• Isolation of cell free or Naked DNA .The cells are broken and the DNA released .The cell
free DNA is subsequently isolated and collected.

Second:
• Mixing of Donor DNA with recipient competent cell. The naked donor DNA is incubated
with the competent recipient cell to which it binds.
Procedure:

Third:

• Uptake and recombination of donor DNA .

• Donor DNA binds to competent recipient cell, following which it enters the recipient
cells.

• Portions of the donor DNA align at random with genes on the recipient of DNA and
segment of the two DNA are exchanged .

• The exchanged inserts Donor genes into the recipient cells DNA.
Application:

Application of Transformation. Transformation is used :

● To make multiple copies of DNA , called DNA cloning .

● To make large amounts of specific human proteins, example human insulin ,which can be
used to treat people with Type I diabetes.

● To genetically modify a bacterium or other cell

REFERENCES:
• FM Ausubel, R Brent, RE Kingston, et al. (eds.) (1999) Introduction of DNA into mammalian cells. Current
Protocols in Molecular Biology, chap. 9. New York, NY: John Wiley.
• Hanahan D, Jessee J and Bloom FR (1991) Plasmid transformation of Escherichia coli and other bacteria. Methods
in Enzymology 204: 63– 113. Harrington JJ, Van Bokkelen G, Mays RW, Gustashaw
• Messing J (1983) New M13 vectors for cloning. Methods in Enzymology Osoegawa K, Yeong Woon P, Zhao B, et
al. (1998) An improved approach for construction of bacterial artificial chromosome libraries. Genomics 52: 1– 8.
• Arena, Christopher B.; Sano, Michael B.; Rossmeisl, John H.; Caldwell, John L.; Garcia, Paulo A.; Rylander,
Marissa Nichole; Davalos, Rafael V. (2011). "High-frequency irreversible electroporation (H-FIRE) for non-thermal
ablation without muscle contraction". Biomedical Engineering Online.
• Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982). 
• "Gene transfer into mouse lyoma cells by electroporation in high electric fields“.
• Garcia PA, Neal RE, Rossmeisl JH, Davalos RV (2010). "Non-thermal irreversible electroporation for deep
intracranial disorders". Conference Proceedings. 2010: 2743–6. d
• ^ Garcia PA, Rossmeisl JH, Neal RE, Ellis TL, Olson JD, Henao-Guerrero N, Robertson J, Davalos RV (July 2010).
"Intracranial nonthermal irreversible electroporation: in vivo analysis". The Journal of Membrane Biology. 236 (1):
127–36. ^ Neal RE, Garcia PA, Rossmeisl JH, Davalos RV (2010). "A study using irreversible electroporation to treat
large, irregular tumors in a canine patient". Conference Proceedings. 2010: 2747–50
• ^ Potter, H (2003). "Transfection by Electroporation". Current Protocols in Molecular Biology.