S2 Biomedik, Biomol, Biomedical Engineering, Genetic Engineering, Budiman

GENETIC
ENGINEERING
Budiman Bela
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Introduction
 This lecture is provided for
Semester 2, Medical Students
 Objective : To introduce the basic
principles of Genetic Engineering
for Medical Students
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TOPICS
 Cloning of genetic materials (DNA

fragments)
 Transfer of genes into living cells
 Lecturer: Budiman Bela, Department
of Microbiology, Medical Faculty
University of Indonesia
 Examples of genetic engineering
application in Medicine
 Ethical aspects of genetic engineering
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 Recombinant DNA technology
 Genetic engineering
 Gene cloning
...are all ways to say essentially the same thing.

They mean:
 isolating desired DNA fragments

 joining them in new combinations and
 …..introducing the newly combined DNA into a
living organism.
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http://www.bio.miami.edu/dana/250/25003_10.html
! E. coli is the most widely used cloning host for amplification
of recombinant DNA
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http://www.bmb.psu.edu/courses/micro106/biotech/biotechover.jpg
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http://www.bmb.psu.edu/courses/micro106/biotech/biotech.htm
 The idea is simple if the practice is not:
select the desired gene (or genes) to be
inserted into the organism
 cut two DNA molecules into fragments with

special (restriction) enzymes
 splice the fragments together in the desired

combination
 introduce the new DNA into a living cell for

replication
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 Early 1970's, Herbert Boyer, Stanley Cohen,
Paul Berg and co-workers  Started
Recombinant DNA Technology:
insertion of foreign pieces of DNA in to host
cells and cloned those host cells to produce
multiple copies of the inserted DNA.
 Currently, there are many sophisticated
techniques available for doing essentially the
same thing: inserting DNA from one species into
another species, and allowing that recipient
species to replicate, producing multiple copies of
the new RECOMBINANT DNA.
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 Some of the Goals of the DNA Technologist:
! 1. Isolation of a particular gene, part of a

gene or region of a genome
! 2. Production of a desired RNA or protein
molecule in large quantities
! 3. Increased production efficiency for
commercially made enzymes and drugs
! 4. Modification of existing organisms so that
they express a particularly desirable trait
not previously encoded in the genome.
!5. Correction of genetic defects in complex
organisms, including humans.
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MAIN CONCEPTS and DEFINITIONS in
RECOMBINANT DNA TECHNOLOGY:
 Recombinant DNA is made by splicing a DNA
fragment of interest into a small, quickly
replicating molecule (such as a bacterial
plasmid). Essentially, you can make huge
numbers of the DNA fragment you want by
sticking it into a very busy piece of DNA.
 An organism containing an artificially inserted,
! foreign piece of DNA is said to be TRANSGENIC.
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How do you get the piece of DNA that you want to
replicate and study?
 The organism from which the DNA of interest is
! extracted is called the DONOR.
 The DNA into which the DNA of interest is
inserted (often a bacterial plasmid) is called a
! VECTOR.
 To excise a piece of DNA from a donor organism,
! RESTRICTION ENZYMES may be used. These

act somewhat like "enzymatic scissors," slicing
through the DNA at specific, recognized
sequences.
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 Once the DNA is excised, DNA ligase is the
"enzymatic glue" used to insert it into replicating
! DNA of the host cell.
 A vector molecule with an insert of foreign DNA is
a RECOMBINANT DNA MOLECULE (sometimes
! called CHIMERIC DNA).
 Vectors are often mixed with bacterial strains
which take them up and incorporate them into
their own genomes (our old pal
! TRANSFORMATION), or simply replicate them
when they replicate their own genomes in
preparation for fission.
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!
 Plating out a bacterial strain carrying your
recombinant DNA vector will allow you to grow a
!
large number of your desired DNA fragment,
which is your DNA CLONE.
!  Once you have a large amount of cloned DNA !

you can characterize the DNA (sequencing; gene
function, etc.), modify it (if desired) and reinsert it
into a recipient (host) organism.
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! TYPE II RESTRICTION ENZYMES recognize and
cut out specific TARGET SEQUENCES on the
DNA. There are different restriction enzymes, and
each one recognizes specific RESTRICTION
SITES (most of which are palindromes) on the
DNA molecule, where it cuts them, making highly
reactive "sticky ends" .
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The vector is also cut open with restriction
enzymes, creating another set of "sticky ends"
where your recombinant DNA fragment can insert
and be "glued in" with DNA ligase.
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 Some restriction enzymes also cut DNA to form "blunt"
ends (without single-stranded tails), which also can be
inserted into target DNA via the action of DNA ligase.
 DNA ligase isn't picky: it can't tell the difference between
foreign and host DNA (who'd figure it would ever have
to?), and this enables the creation of chimeric DNA--
DNA from two separate sources.
 Each enzyme recognizes and cuts specific DNA
sequences. For example, BamHI recognizes the double
stranded sequence:
 5'--GGATCC--3'
3'--CCTAGG--5'
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 To summarize...
 Most restriction enzymes are specific to a single
restriction site
 Restriction sites are recognized no matter where
the DNA came from
 The number of cuts in an organism's DNA made
by a particular restriction enzyme is determined
by the number of restriction sites specific to that
enzyme in that organism's DNA.
 A fragment of DNA produced by a pair of
adjacent cuts is called a RESTRICTION
FRAGMENT.
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 To summarize...
 A particular restriction enzyme will typically cut an
organism's DNA in to many pieces, from several
thousand to more than a million!
 There is a great deal of variation in restriction
sites even within a species.
 Although these variations do not have phenotypic
expression beyond the base sequences
themselves, the variants can be considered
molecular "alleles," and they can be detected
with sequencing techniques.
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 To summarize...
 As such, they can be used in mapping studies
similar to the way true genes with known
phenotypic effects can be used, but skipping the
breeding steps and going straight to the
molecules.
 These "molecular alleles" are a type of
MOLECULAR MARKER, as they can be detected
and located with labeled probes.
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SEPARATING RESTRICTION FRAGMENTS AND
VISUALIZING DNA
 Once the DNA is cleaved, the fragments can be isolated
from one another via electrophoresis, a process by which
molecules of different sizes and chemical/electrical
properties migrate differentially through an electrically
charged gel).
 Here's the play-by-play, should you ever need it...
 agar and buffer are boiled and poured into a mold
 wells are punched into the molten agarose with a toothed
"comb"
 samples are loaded into the wells when agarose
solidifies
 slab is submerged in buffer
 electric current applied to electrodes at opposite ends of
the bath
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SEPARATING RESTRICTION FRAGMENTS AND
VISUALIZING DNA
 DNA fragments migrate towards positive pole (sugar-
phosphate backbones are negatively charged)
 DNA samples are thus sorted by size, with larger
molecules moving more slowly through the agarose
 fragment resolution can be increased by increasing the
agarose concentration, creating smaller "holes" in the
agarose gel
 DNA is stained with ethidium bromide, which intercalates
between base rungs and fluoresce orange under UV light
 DNA fragment lengths can be inferred by comparing their
positions to those of known fragments in a reference gel
 (migration distance is inversely proportional to the
logarithm of fragment length (in base pairs))
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CELL TRANSFORMATION
 You already are familiar with the term
"transformation," which means that a host cell
DNA molecule has taken up and incorporated a
piece of DNA that was not originally a part of it.
 Creating transgenic organisms involves
! transformation of host DNA, not with DNA from
the same species, but with DNA from a
different species.
 Some bacterial species will readily take up
foreign DNA, and are said to be COMPETENT. !
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Transformation
Transformation: DNA picked up
directly from the medium and
recombined into the genome
heteroduplex
! Competent cell:
bacterial cell
capable of picking
up DNA
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CELL TRANSFORMATION
 However, other useful bacterial species can be
"forced" to take up DNA fragments by methods
such as exposure to a salt solution (e.g., calcium
chloride), or heat shocking.
 Transforming eukaryotic cells isn't as simple.
Here are a few methods in use...
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Transfection
 Transfection :
the introduction of foreign material into eukaryotic cells.
! 
Transfection typically involves opening transient pores or
'holes' in the cell plasma membrane, to allow the uptake of
material.
 Genetic material (such as supercoiled plasmid DNA or siRNA
constructs), or even proteins such as antibodies, may be
transfected.
 Transfection is frequently carried out by mixing a cationic lipid
with the material to produce liposomes, which fuse with the
cell plasma membrane and deposit their cargo inside.
 The term transfection is most often used in reference to
! mammalian cells, while the term
 transformation is more often used for the same
process in bacteria and, occasionally, plants.
! !
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Transfection
 Methods of transfection:
 There are various methods of introducing foreign DNA into a
cell.
 One of the cheapest (and least reliable) is transfection by
! calcium phosphate precipitation, originally

discovered by S. Bacchetti and F. L. Graham in 1977.[1]
HEPES-buffered saline solution (HeBS) containing phosphate
ions is combined with a calcium chloride solution containing the
DNA to be transfected. When the two are combined, a fine
precipitate of calcium phosphate will form, binding the DNA to
be transfected on its surface. The suspension of the precipitate
is then added to the cells to be transfected (usually a cell culture
grown in a monolayer). By a process not entirely understood, the
cells take up some of the precipitate, and with it, the DNA.
 Other methods of transfection include:
!  electroporation
 heat shock, and
! !
 proprietary transfection reagents such as Lipofectamine and
Fugene.
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Transfection
 Other methods use highly branched organic compounds,
so-called dendrimers, to bind the DNA and get it into the
cell. A very efficient method is the inclusion of the DNA
to be transfected in liposomes, i.e. small, membrane-
bounded bodies that are in some ways similar to the
structure of a cell and can actually fuse with the cell
membrane, releasing the DNA into the cell. For
eukaryotic cells, lipid-cation based transfection is more
typically used, because the cells are more sensitive.
 A direct approach to transfection is the gene gun, !
where the DNA is coupled to a nanoparticle of an inert
solid (commonly gold) which is then "shot" directly into
the target cell's nucleus  BIOLISTICS
 DNA can also be introduced into cells using viruses as a
carrier. In such cases, the technique is called viral
! transduction, and, the cells are said to be
transduced.
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Stable and transient transfection
 For most applications of transfection, it is sufficient if the
transfected gene is only transiently expressed. Since the DNA
introduced in the transfection process is usually not inserted
into the nuclear genome, the foreign DNA is lost at the later
stage when the cells undergo mitosis. If it is desired that the
transfected gene actually remains in the genome of the cell and
its daughter cells, a stable transfection must occur.
 To accomplish this, another gene is co-transfected, which gives
the cell some selection advantage, such as resistance towards
a certain toxin. Some (very few) of the transfected cells will, by
chance, have inserted the foreign genetic material into their
genome. If the toxin, towards which the co-transfected gene
offers resistance, is then added to the cell culture, only those
few cells with the foreign genes inserted into their genome will
be able to proliferate, while other cells will die. After applying
this selection pressure for some time, only the cells with a
stable transfection remain and can be cultivated further.
!
! A common agent for stable transfection is Geneticin, also

known as G418, which is a toxin that can be neutralized by the
product of the neomycin resistant gene (neo gene).
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! !
ELECTROPORATION
 if host cell has cell walls, enzymes are used to dissolve
the walls, leaving only a protoplast (cell without walls)

 Foreign DNA is introduced via ELECTROPORATION--
protoplasts are exposed to a short electrical pulse which

opens transient membrane channels through which DNA
can pass
 transformed cells can then be cultured in media that
allows re-formation of cell walls and normal growth into a
whole organism (plants, fungi, some protists).
 Animal cells lack cell walls, and so are easily
transformed via electroporation.
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BIOLISTICS
 BIOLISTICS is the process of bombarding cells
! with microscopic projectiles (usually made of an

inert substance such as tungsten or gold) and
coated with DNA
 These are shot at high velocity from a particle
gun into cells or tissue

 This technique is promising for use in live
organisms
 And it was undoubtedly invented by A Guy.
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TRANSDUCTION
 Viruses with affinity for certain cell types can
! also be used as vectors if they are "loaded" with

desired foreign DNA and allowed to infect target
host cells
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MICROINJECTION
 One greatly desired goal is the introduction of genes into
all cells of an animal affected with a genetic disorder, in
the hopes of allowing the faulty cells to transform and
substitute functional genes for faulty ones.
 However, you can't regenerate an entire animal from a
single transformed cell. Instead, an entirely genetically
altered animal can be obtained via MICROINJECTION. !
!  To generate a transgenic animal, foreign DNA
must be inserted into a zygote or very early embryo.
 DNA is injected directly into the nucleus of the cell with an
extremely tiny pipette.
 Once DNA transfer is accomplished, it is sometimes (if the
researcher is lucky!) incorporated into the host cell
chromosome
 The transformed zygote/embryo can then be implanted
into a surrogate mother for growth and development.
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CLONING VECTORS
 Foreign DNA can also be introduced into cells with a
vector, or cloning vehicle. The type of vector depends on
the type of tissue and the task at hand. All
vectors/cloning vehicles, such as a PLASMID for cloning
vector must :
!  have an origin of replication so that endogenote DNA
can be replicated by the host cell's machinery
!  be small, and unlikely to degrade during purification
!  have several unique restriction sites so that the vector
DNA will be cut only in the desired location, and that
several such locations will be available for insertion of
foreign DNA
!  have markers gene (such as antibiotic resistance gene)
that can indicate (in culture) whether transformation has
been successful
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VECTORS FOR GENE DELIVERY
 There are many types of vectors in use and under study
for future use, including...

!  retroviruses
!  adenoviruses
!  adeno-associated viruses (AAV)
 herpes simplex virus
 rhinoviruses
!  Human Immunodeficiency Virus (HIV)
 plasmids of various types (Read the section in your text on
the pBR322 and pUC plasmids. Pay special attention to
the very cool visual trick we can use to isolate successfully
inserted vectors from those that didn't get an insertion into
the polylinker site on the plasmid.)
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Blue and White Colonies
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CLONING VECTORS
 phage lambda
 single-stranded DNA phages (useful because DNA
sequencing is carried out on single-stranded DNA (Sanger
method).
!  cosmids (hybrids of phage lambda and plasmids, and
advantageous because they can be used to insert
relatively large fragments of DNA into a host cell)
 Each has its benefits and drawbacks. The search for the
perfect vector continues--because the perfect vector
probably does not exist. (There's probably no single
vector that will work for every purpose.)
 The overall object: get a vector that will allow you to
clone large amounts of the DNA fragment of interest.
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DNA LIBRARIES
 A restriction fragment is to a DNA library as a
single book is to a regular library: it's only one bit
of a huge compilation of information.
 Every organism has a genome, and
theoretically, we could have a DNA library for
every species. (In your dreams. By the time that
could happen, most species will be extinct
because we've spent so much time trying to
genetically engineer new ones that we will have
lost the ones we already have, and which took
3.5 billion years to evolve...)
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 A DNA LIBRARY is a collection of cloned restriction
fragments from a single organism's genome. The goal is
to have a library containing clones of ALL the organism's
genes. But like real libraries (particularly U.M.'s)--not all
DNA libraries are complete.
 · A GENOMIC LIBRARY is a DNA library containing an
organism's complete genome, in the form of small DNA
fragments (oligonucleotides) representing known genes.
 · The new field of BIOINFORMATICS involves the use
of computers to analyze and store genetic data, such as
the DNA libraries of particular species.
 · A cDNA LIBRARY is a DNA library made up of DNA
clones reconstructed (using reverse transcriptase) from
some of the organism's mRNA molecules.
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EXPRESSION LIBRARIES are made with a cloning vector that
contains the required regularly elements for gene expression, such
as the promoter region.
 In an E. coli expression vector, an E. coli promoter is placed next to
a unique restriction site where DNA of interest can be inserted.
 If successful, insertion of the foreign gene into the correct reading
frame will result in the gene's being transcribed and translated by the
host E. coli cell.
 blotting and treating the culture will allow the protein of interest to
stick to the nitrocellulose filter
 Known antibodies, radioactively labeled, are washed on to the filter
and allowed to glom onto the protein of interest.
 unbound antibodies are then rinsed off
 the filter is set on top of radiograph film, and labeled colonies are
revealed.
 You can now go back to your original plate, slurp up a bit of clone
carrying the desired gene insert, and replicate them to your heart's
delight.
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PROBES
 A GENETIC PROBE is a radioactively labeled, nucleic acid fragment
of known sequence can allow precise location of a particular DNA
sequence in an unknown, single-stranded DNA (single stranded due
to artificial denaturation via heat, etc.) sample by hybridizing with it.
 How do you make a labeled probe? Here's one way:
 1. Select a protein product of interest, and determine aa sequence.

2. From aa sequence, mRNA sequence can be extrapolated, and
mRNA isolated
3. Use reverse transcriptase to manufacture DNA from the isolated
mRNA
4. Supply radioactive nucleotides (usually labeled with 32P) as raw
material for this synthesis
5. denature the DNA-RNA hybrid nucleic acid and you have single-
stranded, radioactive DNA probe that will bind to the DNA that coded
for the mRNA you initially isolated!
6. Clone it!
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(Note that the mRNA lacks introns, but as long as there
are complementary regions of the gene, the probe should
work, even if the introns form "outloopings" in the
probe-DNA hybrid.)
 One important method for isolating and probing

DNA clones in this way is the SOUTHERN BLOT
TECHNIQUE. This, too, you might get to do in
lab. (Side note: an AUTORADIOGRAPH is a
photograph in which the image on the film has
been formed by exposure to radioactivity.)
 NORTHERN BLOT: Similar technique, but used
to assay RNA.
 WESTERN BLOT: Again, similar, but used to
assay proteins via antibody binding.
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FUNCTIONAL COMPLEMENTATION
 Specific genes can be located for cloning through their ability to
restore wild type phenotype to a mutant organism. Here's how it
works...
 Select a protein of interest, and a wild type organism (Let's say
"Species X" that produced its normal product.
 Create a DNA library from that wild type organism, using an
appropriate vector (bacteria or phages)
 Take library samples and introduce them to mutant colonies of
Species X which cannot produce the protein of interest.
 Plate out your (hopefully) transformed Species X, and select only the
ones that exhibit the wild type phenotype. Those are the ones that
have been successfully transformed.
 Use the transformed colonies to recover and clone the wild type
gene, which you know is present because its product is being
manufactured.
 Remember: the vectors are supplying only a fragment of the Species
X genome, which is why this works!
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 Vectors do not always insert exactly where desired! In
fact, there are three general scenarios that may occur if
donor DNA is actually incorporated into a recipient cell.
 And in many instances, the DNA of interest isn't
accepted at all. Them's the breaks.
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POSITIONAL CLONING
 As you might have surmised, it takes a lot of time and
work to locate a gene of interest in a library full of

unknown clones. One way to lighten this burden is to
incorporate information about a particular gene's actual,
physical location in the genome.
 Both probing and complementation can be used in
positional cloning.
 CHROMOSOME WALKING starts with a known gene
that is linked to a particular unknown gene of interest. By

gradually hybridizing, fragmenting and re-cloning, the
chromosome gene order can be gradually reconstructed.
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CHROMOSOME WALKING
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DNA SEQUENCING
 If you have grown a DNA clone of interest, but do not know its base
sequence, the next important step is to determine the sequence of
your cloned fragment.
 There are many different methods used for DNA sequencing,
including
 Dideoxy Method
 Your assignment is to study the three diagrams and understand the

general workings of this protocol. However, the best way to learn it is
to actually DO it, which is why you might want to consider taking BIL
251. (Shameless plug!)
 The Main Idea: Once you have completed your dideoxy binding and
made your Sanger sequencing gel (via electrophoresis), you will
have a huge number of DNA fragments, each one nucleotide longer
than the last. The nucleotide sequence can essentially be read
directly from the gel.
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DNA SEQUENCING
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DNA SEQUENCING http://www.bio.miami.edu/dana/250/25003_10.html
DNA SEQUENCING
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APPLICATIONS OF RECOMBINANT DNA
TECHNOLOGY: PANDORA'S BOX?
 IN VITRO MUTAGENESIS
 If the sequence of a wild type gene is known...
 a short, complementary sequence known as an
OLIGONUCLEOTIDE can be made
 a site-specific mutation can be induced in the
oligonucleotide
 the oligonucleotide can contain a mutation of any desired
type, including base pair substitution, insertion, deletion,
etc.
 when taken up by a phage vector (usually phage M13), it
can then be inserted into bacteria for cloning and further
study.
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REVERSE GENETICS
 In the Good Old Days, an investigator would discover an

organism with a mutant phenotype, determine that it had
a particular mutant allele, determine the DNA sequence
for that allele and then infer the amino acid sequence of
the faulty protein. Today...
 a protein or a gene of unknown function is isolated.
 an ORF ("open reading frame") is a segment of DNA flanked
by a start and stop codon. Though it has been sequenced
and found, its function and product are not known. It is a
putative gene.
 To determine the function of the ORF, a site-specific mutation
can be induced (as described above)
 This can then be radioactively labeled, inserted into a vector
and cloned.
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REVERSE GENETICS
 The radioactive clones can be used as probes to find the

relevant gene.
 Insertion of the mutant sequence into the genome of a
bacterium can be used to determine the function of the
disrupted gene, since the bacterium will be unable to
manufacture the product of the disrupted gene.
 This works because a eukaryote gene inserted into a bacterium
will always be expressed (whether wild type or mutant), since
the bacterium lacks the eukaryotic gene's regulatory sequences
that will turn it on or off.
 This technology is important in GENE KNOCKOUT, which we'll
discuss shortly.
 Also, genetically engineered bacteria (and fungi) like this can
be used to produce a eukaryotic gene product in great
quantities--a commercial boon! These "designer bacteria" are
possibly even subject to patents.
(Does The Creator Person get a royalty?)
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EUKARYOTIC VECTORS
 For certain purposes, plasmid DNA and bacterial vectors
are not sufficient. For example, E. coli lacks some of the

enzyme systems that allow post-transcriptional/post-
translational modification of proteins.
Also, if you want to study the function of the eukaryotic
genome in vivo, the only way to do so is to work with
eukaryotic cells.
And finally, if we want to manipulate eukaryote genes for
medical and economic reasons, we have to use
eukaryotic vectors.
 What to do? There are several eukaryotic vectors in use.
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EUKARYOTIC VECTORS
 YEAST ARTIFICIAL CHROMOSOME
 In nature, Baker's yeast (Sacchromyces cerevisieae) contain a small

plasmid. However, the inclusion of the plasmid is unstable, and the
wee thing tends to get lost as the yeast divide.
 To solve this problem, geneticists have succeeded in manufacturing
an artificial version of this plasmid by inserting into a bacterial
plasmid
 a yeast centromere
 the yeast origin of DNA replication (known as ARS--autonomously

replicating sequence).
 sometimes, telomeric sequences (easy cutting spots for making
linear chromosomes out of the circular plasmid)
 This modified plasmid is called a YEAST ARTIFICIAL !
CHROMOSOME (YAC). It can carry very large pieces of DNA (from
a donor eukaryote) to be inserted into eukaryotic cells. (Note that
whereas a cosmid can carry only about 50 kb, a YAC can carry as
much as 800 kb!).
!
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EUKARYOTIC VECTORS
 VECTORS SPECIFIC TO ANIMALS...
 DNA tumor virus SV40 (simian vacuolating virus)

- This can transform normal eukaryote cells into
cancer cells.
 · SV40 can carry foreign DNA.
 · Can be replicated in eukaryotic cells for
cloning of carried DNA

 Many other viruses with an affinity for animal
cells can be used, as we already mentioned.
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EUKARYOTIC VECTORS
 VECTORS SPECIFIC TO PLANTS...
 A soil bacterium known as Agrobacterium

tumefaciens causes gall tumors in dicot plants.
The causative agent of the tumors is a plasmid
named Ti, which transforms the normal plant cell
into a cancerous cell when it is integrated into
the plant's DNA. (OO! Hint that this will be a
good vector!).
 Geneticists can insert foreign DNA into this
plasmid, and use its affinity for dicot plant DNA
to facilitate insertion of new genes into plants.
 function or disorder.
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EUKARYOTIC VECTORS
 When foreign DNA is inserted into a eukaryotic
genome, TRANSFECTION is said to have
occurred (similar to bacterial transformation, but
renamed to distinguish it from eukaryotic
"transformation" which generally refers to a cell's
becoming cancerous). A eukaryotic organism
which has taken up foreign DNA with a vector is
said to be TRANSGENIC. And as you'll see,
transgenics transcends taxonomic relationships.
Very distantly related organisms can be artifically
implanted with each other's DNA. Scary!
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EUKARYOTIC VECTORS
 A few examples...
 Transgenic mice are routinely made by injecting vectors

containing cloned DNA into oocytes or even one- or two-
celled embryos which are then re-implanted.
 Note that only in about 15% of these operations is the
foreign DNA actually incorporated into the host's
genome. It doesn't always work!
 The first transgenic eukaryote ever made (1988) was a
mouse that carried a gene that predisposed it to cancer.
It is used as a model to study cancer. (Yes, someone
tried to patent this mouse, and there's a lot of argument
as to whether genetically engineered higher organisms
should be patentable!)
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EUKARYOTIC VECTORS
A few examples...
 Since it's sometimes difficult to determine whether an inserted gene
! is "turned on" (more on this when we do gene expression next

week), geneticists sometimes will insert a REPORTER GENE !
alongside the gene of interest. Such a reporter gene is one which is
easily detectable in phenotype. When it is functioning, its product's
presence is a good indication that the adjacent gene of interest is
also working.
 One such reporter gene that gives rather spectacular results is the
LUCIFERASE gene, which causes the transgenic organism
expressing it to bioluminesce. When you see pictures of glowing
mouse embryos or glowing tobacco plants, you're seeing the results
of a genetic marker that's used to detect the possible activity of the
gene in its vicinity that's really the one of interest.
 The race is on. There are transgenic organisms of many kinds, most
created in order to study a particular human function or disorder.
11/10/2019 73
11/10/2019 74
 Transgenic animals are becoming
practically commonplace.
 transfection accomplished at zygote stage
(affects all future generations)
 transfection accomplished in target cells
(affects only the individual; not future
generations) This dichotomy is at the root
of the future of human gene therapy. If we
alter human disease genes, do we plan to
do it at the zygote stage--or the somatic
stage? Huge bioethical implications!
11/10/2019 75
Transfection at Zygote Stage
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 What on earth is a KNOCKOUT MOUSE?
 Mice have been used extensively in transgenic studies,
and are often the organism of choice for producing
clones of DNA in which a specific gene has been
targeted and inactivated ("knocked out") In most cases, a
gene transfected into a mouse cell is randomly
incorporated into the genome. We just don't know exactly
where it goes.
*Once in a while, the donor gene completely replaces the
mouse locus where it inserts (this happens if the foreign
gene lines up with its homologue before crossing over,
and is taken up instead of the true homolog's locus).
*This property has allowed the creation of a new
KNOCKOUT technology.
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Knocking Out a Gene
11/10/2019 78
 *To make a KNOCKOUT MOUSE, the geneticist
transfects the normal mouse oocyte with a gene that is a
defective version of the one s/he wishes to study. That
is, the normal gene is "knocked out" by the mutant gene
carried by the vector.(Note that the rate of successful
transfection is pretty low--about 15%)
*If a successfully transfected oocyte is fertilized and
grows into a new mouse, that mouse will be
heterozygous for the mutant gene.
*Breeding two such heterozygotes together should give
you 25% homozygous recessives.
*By studying these homozygotes, the scientist can
determine whether the gene in question is essential, or
what its normal functions are by noting the deficiencies in
the knockout mice.
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Knockout Mouse
11/10/2019 80
Knockout Mouse
11/10/2019 81
Kloning materi genetik
(gen)
 Kloning materi genetik pada umumnya
dilakukan dengan menyisipkan materi
genetik terpilih (gene of interest) ke dalam
DNA sirkuler yang dapat bereplikasi
secara tersendiri, tanpa mengikuti siklus
replikasi sel
 PLASMID
 DNA sirkuler yang digunakan sebagai

rangka dasar (backbone) dalam proses
kloning biasanya diperbanyak dalam
sistem prokariota
(Escherichia coli  E. coli)
11/10/2019 82
(gen)
 PLASMID: materi genetik sirkuler
yang dapat bereplikasi sendiri.
Dapat ditemukan pada sel ragi
(eukariot) dan pada sel bakteri
(prokariota)
 Agar dapat bereplikasi sendiri,

plasmid memerlukan susunan
nukleotida (sekuens nukleotida)
tertentu: ori (origin of replication)
11/10/2019 83
(gen)
 Untuk memperbanyak DNA yang
terdapat pada plasmid, plasmid
harus dimasukkan ke dalam E. coli
yang masih hidup, melalui proses
yang disebut Transformasi
 Transformasi E. coli oleh DNA

plasmid (DNA asing) dapat
dilakukan dengan berbagai teknik
kimiawi (contoh: teknik Kalsium
Klorida, dsb) maupun elektroporasi
11/10/2019 84
(gen)
 Penyisipan gen ke dalam Plasmid dilakukan dengan
bantuan enzim endonuklease restriksi (restriction
endonuclease = restriction enzymes)
 Segmen DNA (gen) yang akan disisipkan ke dalam
plasmid harus diapit oleh susunan nukleotida (situs
restriksi) yang dapat dikenali oleh enzim restriksi yang
akan digunakan dalam proses kloning
 Penentuan enzim restriksi yang akan digunakan dalam
proses kloning dapat dilakukan berdasarkan situs restriksi
yang terdapat pada vektor Plasmid yang tersedia, atau
sebaliknya, yaitu ditentukan lebih dahulu situs restriksi
yang akan digunakan untuk proses kloning, kemudian
situs restriksi tersebut diciptakan pada Plasmid dan pada
kedua ujung Segmen DNA yang akan disisipkan (DNA
sisipan = Insert DNA)
11/10/2019 85
Transformation
Transformation: DNA picked up
directly from the medium and
recombined into the genome
heteroduplex
Competent cell:
capable of picking
up DNA
11/10/2019 86
Transduction
Viral products produced, host

genome fragmented
~1 phage/10,000 will
pick up chromosomal
DNA...
11/10/2019 87
Plasmids
11/10/2019 88
Conjugation
11/10/2019 89
Resistance Plasmid
11/10/2019
90
Genetic Engineerin
11/10/2019
91
Restriction Endonucleases
• A Restriction Endonucleases will cut both
strands of a DNA duplex at a specific place
• These “places” need not be directly opposite:
5’…GAATTC…3’
3’…CTTAAG…5’
5’…G -OH
5’…G 5’…GAATTC…3’
-OH AATTC…3’
P-AATTC…3’
P-
3’…CTTAA
3’…CTTAAG…5’
3’…CTTAA -P
-P G…5’
HO-G…5’
HO-
• Note that this enzyme used is EcoRI, the first

restriction endonuclease characterized
11/10/2019 92
Sticky Ends
(Ujung Kohesif)
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93
Ligation
11/10/2019 94
Enzyme Sequence Product
EcoRI GÂATTC 5’ sticky ends
BamHI G^GATCC 5’ sticky ends
More RE Enzymes
Bg1II A^GATCT 5’ sticky ends
PvuI CGATC^G 3’ sticky ends
PvuII CAG^CTG Blunt end
MboI GÂTC 5’ sticky ends
HindIII AÂGCTT 5’ sticky ends
HinfI GÂNTC 5’ sticky ends
Sau3A GÂTC 5’ sticky ends
AluI AG^CT Blunt end
TaqI T^CGA 5’ sticky ends
HaeIII G^GCC 5’ sticky ends
NofI GC^GGCCGC 5’ sticky ends
11/10/2019 95
Most ER Recognition
Sequences are Palindromes
GÂATT-C G^GATC-C
C-TTAA^G C-CTAG^G
Nodeba Bob Abedon
A^GATC-C GC^GGCC-GC
T-CTAG^G CG-CCGG^CG
11/10/2019 96
(gen)
 Selain mempergunakan enzim restriksi
yang membentuk ujung kohesif (sticky
ends), kloning juga dapat dilakukan
dengan enzim yang hasil
pemotongannya membentuk ujung
tumpul (blunt end), tetapi efisiensi proses
ligasi DNA pada ujung tumpul tidak
sebaik pada ujung kohesif  lebih sulit
memperoleh plasmid rekombinan
 Rekombinan: gabungan antara dua
segmen DNA: DNA plasmid dan DNA
sisipan)
11/10/2019 97
(gen)
 Alternatif lain yang dapat dilakukan untuk
meningkatkan efisiensi ligasi bila tidak
dapat digunakan enzim restriksi dengan
ujung kohesif adalah dengan
menggunakan Kloning-TA (TA Cloning)
 Dna sisipan pada Kloning-TA pada
umumnya merupakan produk PCR yang
menggunakan enzim polimerasa Taq:
memiliki kecenderungan untuk
menambahkan Adenin (A) yang
menggantung pada ujung 3’
11/10/2019 98
(gen)
 Dna plasmid (vektor) pada

Kloning-TA diperoleh dengan
pemotongan secara tumpul dan
diikuti dengan penambahan ujung
T yang menggantung dengan jalan
mereaksikan ujung 3’ DNA yang
tumpul dengan dTTP dengan
katalisator enzim polimerasa Taq
11/10/2019 99
Kloning TA
5’…CAGCTG…3’
3’…GTCGAC…5’
5’…CAG-OH P-CTG…3’
3’…GTC -P HO-GAC…5’
5’…G -OH5’…GAATTC…3’
P-AATTC…3’
3’…CTTAA3’…CTTAAG…5’
-P HO-G…5’
• Note that this enzyme used is EcoRI, the first

restriction endonuclease characterized
11/10/2019 100
Complementary (c)DNA Note that the reverse
transcriptase is primed
by the poly-A found at
the end of most
eukaryotic mRNAs
Note that there are
issues (& variation) as
to how one makes the
second DNA strand
Here that strand is
generated using a
second enzyme that is
primed via generation
of a hairpin of DNA by
reverse transcriptase
11/10/2019 101
Ligation into Plasmid
Upon Ligation we now have

Recombinant DNA!
11/10/2019 102
Note that
plasmid is vector
Transformation
that carries DNA

into recipient
cells
Other vectors
include viruses
(transduction) as
well as
otherwise inert
projectiles
11/10/2019 103
Virus (Phage) Vector
11/10/2019
104

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S2 Biomedik, Biomol, Biomedical Engineering, Genetic Engineering, Budiman

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GENETIC

 Cloning of genetic materials (DNA

...are all ways to say essentially the same thing.

 isolating desired DNA fragments

 cut two DNA molecules into fragments with

 splice the fragments together in the desired

 introduce the new DNA into a living cell for

! 1. Isolation of a particular gene, part of a

! foreign piece of DNA is said to be TRANSGENIC.

! RESTRICTION ENZYMES may be used. These

!  Once you have a large amount of cloned DNA !

Here are a few methods in use...

! calcium phosphate precipitation, originally

the walls, leaving only a protoplast (cell without walls)

protoplasts are exposed to a short electrical pulse which

transformed via electroporation.

! with microscopic projectiles (usually made of an

gun into cells or tissue

! also be used as vectors if they are "loaded" with

for future use, including...

 1. Select a protein product of interest, and determine aa sequence.

 One important method for isolating and probing

work to locate a gene of interest in a library full of

that is linked to a particular unknown gene of interest. By

 Your assignment is to study the three diagrams and understand the

 In the Good Old Days, an investigator would discover an

 The radioactive clones can be used as probes to find the

are not sufficient. For example, E. coli lacks some of the

 In nature, Baker's yeast (Sacchromyces cerevisieae) contain a small

 the yeast origin of DNA replication (known as ARS--autonomously

 DNA tumor virus SV40 (simian vacuolating virus)

 · Can be replicated in eukaryotic cells for

cloning of carried DNA

cells can be used, as we already mentioned.

 A soil bacterium known as Agrobacterium

 Transgenic mice are routinely made by injecting vectors

! is "turned on" (more on this when we do gene expression next

 DNA sirkuler yang digunakan sebagai

 Agar dapat bereplikasi sendiri,

 Transformasi E. coli oleh DNA

Viral products produced, host

• Note that this enzyme used is EcoRI, the first

 Dna plasmid (vektor) pada

• Note that this enzyme used is EcoRI, the first

Upon Ligation we now have

that carries DNA

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