Lecture 11A Modul Biologi Molekular, Genetic Engineering, FKUI Semester II (MARKED!!) 2009

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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.

http://www.bio.miami.edu/dana/250/25003_10.html

04/11/23 5http://www.bmb.psu.edu/courses/micro106/biotech/biotechover.jpg

! E. coli is the most widely used cloning host for amplification of recombinant DNA

04/11/23 6http://www.bmb.psu.edu/courses/micro106/biotech/biotech.htm

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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


04/11/23 8http://www.bio.miami.edu/dana/250/25003_10.html

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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 genome2. Production of a desired RNA or protein molecule in large quantities3. Increased production efficiency for commercially made enzymes and drugs4. 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:electroporationheat 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

http://www.cas.vanderbilt.edu/bsci111a/recom-2/supplemental.htm

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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 isolated3. Use reverse transcriptase to manufacture DNA from the isolated mRNA4. Supply radioactive nucleotides (usually labeled with 32P) as raw material for this synthesis5. 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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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.


(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.)

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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.


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.


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 VECTORSA 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.

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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!


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.


Knocking Out a Gene

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*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.


Knockout Mouse

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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)

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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)

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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

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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)

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Transformation

Transformation: DNA picked up directly from the medium and recombined into the genome

heteroduplex

Competent cell: capable of picking up DNA

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Transduction

Viral products produced, host genome fragmented

~1 phage/10,000 will pick up chromosomal DNA...

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Pla

smi

ds

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Conjugation

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Res

ista

nce

P

lasm

ids

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Gen

etic

E

ng

inee

rin

g

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Restriction Endonucleases

• A Restriction Endonucleases will cut both strands of a DNA duplex at a specific place

• These “places” need not be directly opposite:

• Note that this enzyme used is EcoRI, the first restriction endonuclease characterized

5’…GAATTC…3’ 3’…CTTAAG…5’


5’…G -OH P-AATTC…3’ 3’…CTTAA -P HO-G…5’


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Sti

cky

En

ds

(Uju

ng

Ko

hes

if)

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Lig

atio

n

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Mo

re R

E E

nzy

mes

Enzyme Sequence Product

EcoRI GÂATTC 5’ sticky ends

BamHI G^GATCC 5’ sticky ends

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

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Most ER RecognitionSequences are Palindromes

G^AATT-CC-TTAA^G

G^GATC-CC-CTAG^G

A^GATC-CT-CTAG^G

GC^GGCC-GCCG-CCGG^CG

Nodeba Bob Abedon

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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)

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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’

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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

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Kloning TA

• Note that this enzyme used is EcoRI, the first restriction endonuclease characterized

5’…CAGCTG…3’ 3’…GTCGAC…5’



5’…CAG-OH P-CTG…3’ 3’…GTC -P HO-GAC…5’

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Co

mp

lem

enta

ry (

c)D

NA 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

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Lig

atio

n in

to P

lasm

id

Upon Ligation we now have Recombinant DNA!

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Tra

nsf

orm

atio

nNote that

plasmid is vector that carries DNA

into recipient cells

Other vectors include viruses

(transduction) as well as

otherwise inert projectiles

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Vir

us

(Ph

age)

Vec

tor

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Details about this topic Supporting information and

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Lecture 11A Modul Biologi Molekular, Genetic Engineering, FKUI Semester II (MARKED!!) 2009

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Transcript of Lecture 11A Modul Biologi Molekular, Genetic Engineering, FKUI Semester II (MARKED!!) 2009