plasmids
A plasmid is a circular, self-replicating DNA molecule carrying a few, useful but non necessary genes.
Occurence
procaryote organisms
eukaryotic organisms like Entamoeba histolytica, yeast etc.
Their size varies from 1 kbp to over 400 kilobase pairs (kbp).
In a single cell there are anywhere from
one copy, for large plasmids,
to hundreds of copies of the same plasmid.
We speaks of low and high copy number plasmids
Plasmids are easy to manipulate and isolate from bacteria (kits).
After being modified, they can be integrated into other genomes, plants, protists, mammals,
thereby conferring to other organisms whatever genetic functionality they carry.
Thus, this gives the ability to introduce genes into a given organism by using bacteria to
amplify the hybrid genes that are created in vitro.
This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.
------------------------to carry: llevar
a few: un poco
size: tamaño
anywhere: en cualquier parte.
whatever: cualquier -a
Bacterial plasmid
types
Plasmids are classified
1. by their ability to be transferred to other bacteria
Conjugative
The sexual transfer of plasmids to another bacterium through a pilus. those plasmids possess
the 25 genes required for transfert
Non-conjugative
Non-conjugative plasmids don’t initiate conjugaison.
They can only be transferred with the help of conjugative plasmids.
mobilisable
An intermediate class of plasmids are mobilisable, and carry only a subset of the genes
required for transfer. These plasmids can 'parasitise' another plasmid, transferring at high frequency
in the presence of a conjugative plasmid
incompatibility groups:
Several types of plasmids could coexist in a single cell.
On the other hand, related plasmids are often 'incompatible', resulting in the loss of one of them
from the cell line.
---------------------------to classify: clasificar
ability: aptitud, capacidad
a subset is a set whose members are members of another set.
conjugative plasmids
The sexual transfer of plasmids to another bacterium through a pilus. Those plasmids, plasmidos F,
possess the 25 genes required for transfert.
---------------------------El plásmido F contiene the 25 genes que controlan la producción de los pilis.
Los pili se observan prácticamente solo en bacterias Gram negativas.
transfert, m ¿ Cuál palabra escoger?
in: They possess the genes required for transfert
1.
(de prisionero, de población) traslado.
2.
(de fondos, de mercancías) transferencia.
3.
(de negocio) traspaso.
4.
(de bienes inmuebles) transmisión.
5.
Psicol transferencia
rolling circle replication:
passes a plasmid through a pilus. A nice film showing how it works can be seen at:
http://info.bio.cmu.edu/Courses/03441/TermPapers/2000TermPapers/group2/fig3.html
What’s important in rolling circle replication is to find out the two 3’ ends used in the process
2. by function
1. Fertility-(F) plasmids,
They are capable of conjugation (they contains the genes for the pili).
2. Resistance-(R) plasmids,
contain gene (s) that can build resistance against one or several antibiotics or poisons.
3. Col-plasmids,
contain genes coding for colicines, proteins that can kill other bacteria.
4. Degradative plasmids,
able to digest unusual substances, e.g., toluene or salicylic acid.
5. Virulence plasmids,
turn a bacterium into a pathogen.
6. addiction system.
These plasmids produce both a long-lived poison and a short-lived antidote.
Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails
to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison
from the parent cell.
--------------------------------addiction: dependencia, adicción
daughter: hija
to inherit: heredar.
lingering: persistente
Episomes
are units of genetic material composed of a series of genes that sometimes has an
independent existence in a host cell and at other times is integrated into a chromosome of the cell,
replicating itself along with the chromosome.
Episomes exist in all organisms.
In bacteria, episomes are:
viruses:
Bacteriophages in bacteria (lytic or lysogenic phages, prophages and proviruses)
Sex factors Hfr:
sex factors like the F factor F+ is autonomous, extrachromosomal.
Hfr (or high frequency recombination) is the same sequence, but integrated into the host chromosome.
Plasmids:
can be integrated into a chromosome.
transposons:
Transposons are known as mobile genetic elements (they were discoverd in maize
by Barbara McClintock*)
While they can also exist outside of the chromosome, they prefer to, and are designed, to integrate
into the chromosome following their movement from one cell to another.
For example, Class 1 transposons encode drug resistance genes.
-------------------------host: huésped
host cell, célula huésped
Integrons are mobile DNA elements able to capture genes, notably those encoding antibiotic resistance,
by site-specific recombination.
Class 1 integrons have been examined the most extensively. They consist of a variable region bordered
by 5' and 3' conserved regions. The 5' region is made up of the int1 gene, attI, and the promoter Pr . Pr
drives transciption of genes within the variable region. The 3' region consists of qacED1*, coding a
multiple substrates efflux pump, a sulfonamide resistance gene.
The antibiotic resistance genes that integrons capture are located on gene cassettes. The cassettes
consist of a promoterless gene and a recombination site (attC). The cassettes can exist as free, circular
DNA but cannot be replicated or transcribed in this form. A recombination event occurs between attI and
attC, integrating the cassette into the integron. The gene on the cassette is then bound by the attI site on
the 5' side and by attC on the 3' side.
*http://www.biochemj.org/bj/376/0313/bj3760313.htm
conformation
In bacteria, plamids are found in the supercoil (superenrollado) form.
Supercoil s and relaxed (relajado) forms can be seen under the electron microscope,
in electrophoresis or after centrifugation.
The supercoiled form migrate and sediments quicky in in eletrophoresis and centrifugation
respectively.
Topoisomerases help to pass from one form to the other as we saw it earlier.
Plasmids in molecular biology
Minimum requirements for plasmids useful for recombination technology:
1. Origin of replication (ORI). ORI enables a plasmid DNA to be duplicated
independently from the chromosome
2. Selectable marker: allow to select for cells that have your plasmids.
3. Restriction enzyme sites in non-essential regions of the plasmid.
Plasmid replication initiates in a cis-site called ori. It proceeds either by a rolling circle or
a theta replication mechanism. Some of the plasmid-encoded elements required for their replication,
such antisense RNA molecules and DNA repeated sequences located close to ori,
determine plasmid attributes like copy number and incompatibility.
Plasmids often contain genes or gene-cassettes that confer a selective advantage when they
are inside a bacterium:
resistance to antibiotics
resistance to herbicides
insecticide production
to the bacterium harboring them, for example, the ability to make the bacterium antibiotic resistant.
f1 origin
The only difference between these two vectors is the orientaion of the f1 ori
Either from right to left
Or from left to right
What is the f1 ori
It is the origin of replication of the phage F1
Then, the vectors pGEM includes a bacteriophage f1 origin of replication:
pGEM 3Zf1 (+) in one sens
pGEM 3Zf1 (-) in the other sens.
Then, these vectors can be used for the production of single-stranded
DNA (ssDNA) phages which were used for DNA sequencing.
Each vector is used for the production of one strand.
For induction of ssDNA, bacterial cells containing a pGEM-Zf recombinant are
infected with an appropriate helper phage. The plasmid enters the f1 replication
mode, and the resulting ssDNA is exported from the cells as an encapsidated
virus particle. These particles were used for DNA sequencing.
pGEM-3Zf(+) and pGEM-3Zf(-) vectors share a common backbone and a multiple
cloning site within lacZ sequences flanked by SP6 and T7 RNA polymerase promoters.
These vectors differ only by which DNA strand is packaged with the addition
of helper phage.
For induction of ssDNA, bacterial cells containing a pGEM-Zf recombinant are
infected with an appropriate helper phage. The plasmid enters the f1 replication
mode, and the resulting ssDNA is exported from the cells as an encapsidated
virus particle.
---------------------------------Strand: hebra, cadena.
backbone: la columna vertebral
to flank: flanquear
Tranformation*
In bacteria and yeast:
transformation is the transfer of genetic information from a donor to a recipient
using naked DNA without cell to cell contact. The DNA from the donor is in the
medium where it was secreted. The recipient takes this DNA up:
there is no requirement for cell to cell contact.
The only limitation of this uptake is the restiction system
1. natural transformation (rare but dangerous):
Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from
their surrounding. While naturally transformable bacterial strains exist, these are sufficiently
rare that induced transformation is more important to the geneticist.
A captured DNA has to face restriction!
2. experimental: in the lab, DNA is introduced by one of the following methods:
a. Calcium chloride (followed by heat shock) method
b. Electroporation.
c. Liposomes
---------------------------------------*in eucaryotes other than yeast, transformation is called transfection
In vitro transformation is made during rapid growth.
DNA transformation is a process by which DNA can be transferred into yeast or
bacterias. During rapid growth of E. Coli, a bacterium, the cell membrane has
hundreds of pores, called adhesion zones. Transformation is made during this
Rapid growth, i. e. when these pores are present.
calcium choride
Use E. coli after rapid growth*.
1. In a microtube, gently mix the plasmid DNA with the bacteria as you add it with a pipet.
Incubate the mixture on ice for 20 minutes.
2. Heat shock the mixture by submitting it for 2 minutes at 42°C
Transfer the bacterial suspension to 10 ml of L Broth without antibiotic.
Shake for 45 minutes in a 37°C incubator.
3. Centrifuge the cells at 2200 rpm for 15-20 minutes at 4°C. Discard the supernatant.
4. Resuspend the bacteria in 100 µl of L broth (+ antibiotic) per Petri dish, and plate on
a LB agar + antibiotic plates using the turntable and a sterile glass rod.
5. Determine the transformation efficiency the next day by counting the number of colonies
----------------------------------* It works better when bacterias are first treated with Calcium Chloride.
electroporation
One way of physically introducing DNA into a cell is electroporation.
The diagram shows an electrical circuit for a simple electroporation device.
A solution of DNA fragments containing the gene of interest is added
to the cuvette.
The capacitor is charged by closing the right-hand switch. When the capacitor has
been charged, the direct current pulse is discharged in the cuvette suspension
by closing the left-hand switch.
The DC pulse is thought both to disrupt temporarily the membrane and to
“electrophorese” DNA into cells.
----------------------------a switch: un interruptor
capacitor : nm condensador
reporter: aquí: un testigo.
to close: cerrar
pulse: impulse, descarga
direct current (DC) : nf corriente continua
Electroporation System
Introduce DNA, RNA, and proteins into mammalian, plant, or bacterial cells.
Most system come with the Pulse Control unit, Chamber Safe with a Chamber Rack,
Electroporation Chambers, and a manual.
Equipped with an internal power supply to deliver a range of reproducible electrical
pulses to the cells by capacitor discharge.
Several capacitors provide pulse lengths from <0.1 to >200 ms.
Disposable electroporation chambers are available with a 0.4-cm inter-electrode gap
(standard design) for transfecting mammalian or plant cells, or with a 0.15-cm inter-electrode
gap (microelectroporation design) for transforming yeast or bacterial cells.
liposomes
To physically introduce DNA into cells you can use liposomes.
Liposomes are lipid-bilayer bounded vesicles. Produced by hydrating lipids
in aqueous solutions. If DNA is present in the solution, it becomes incorporated into
the liposomes.
Liposomes interact with cell membranes. The liposomal contents is
transferred to the inside of the cell. Both membrane fusion and endocytosis have
been implicated as mechanisms.
Genes present in the transferred DNA can be expressed transiently.
The transferred DNA may also integrate into chromosomes and
cell lines containing the integrated gene may be selected.
recombinant plasmids
T7 promoter
Eco RI
Hind III
5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagttcacctaaat 3’
3’ acattatgctgagtgatatcccgcttaagctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcgaactcataagatatcaagtggattta 5’
SP6 promoter
T7 and SP6 are (trans) RNA Pol from T7 and SP6 phages.
They bound to to their cis promoters respectively and start making RNA. These RNA can ve used to make probes,
used for micro-arrays or Southern blots.
(In between the T7 and SP6 promoter is the pUC 18 polylinker):
At the left of the molecule is the ATG codon of the LacZ gene.
The native nucleotide sequence of this plasmid is in small caps.
A 54 nt fragment (large caps) interupts but doesn’t disrupt the reading frame
54 is a multiple of 3 !
In the pGEM-3Z plasmid, in addition to the 54 nucléotide, Two promoters were
Also added.
T7 promoter
Eco RI
Hind III
5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagttcacctaaat 3’
3’ acattatgctgagtgatatcccgcttaagctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcgaactcataagatatcaagtggattta 5’
SP6 promoter
opening of the plasmid polylinker: making room for the insert
You have to buy the plasmid, the restriction enzymes and their buffer.
Eco RI
Hind III
5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagttcacctaaat 3’
3’ acattatgctgagtgatatcccgcttaagctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcgaactcataagatatcaagtggattta 5’
+ Eco RI and Hind III
5’ aattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgca 3’
3’ gctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcga 5’
51 bp fragment to be eliminated, by electrophoresis for example
+
5’ tgtaatacgactcactatagggcg
3’ acattatgctgagtgatatcccgcttaa
agcttgagtattctatagttcacctaaat 3’
actcataagatatcaagtggattta 5’
<-------------- place for an insert ------------>
The 51 bp fragment between Eco RI and Hind III is eliminated, by electrophoresis
The opened plasmid (2692 bp) is extracted from the gel either using agarase or
an extraction kit. In each case, you should make the gel with LMP agarose.
LMP means low melting point.
Trimming of the fragment we wanted to clone:
Eco RI
Hind III
5‘ tatctggaattccctgccagtattatatgctgaattcagagattaagccaagcttgtgta 3‘
3‘ atagaccttaagggacggtcataatatacgacttaagtctctaattcggttggaactctat 5‘
+ Eco RI and Hind III
5‘
3‘
aattccctgccagtattatatgctgaattcagagattaagcca
gggacggtcataatatacgacttaagtctctaattcggttgga
3‘
5‘
insertion of the fragment into the open plasmid
5‘aattccctgccagtattatatgctgaattcagagattaagcca
3‘
3‘
gggacggtcataatatacgacttaagtctctaattcggttgga 5‘
+
5’ tgtaatacgactcactatagggcg
3’ acattatgctgagtgatatcccgcttaa
agcttgagtattctatagttcacctaaat 3’
actcataagatatcaagtggattta 5’
5’ tgtaatacgactcactatagggcg aattccctgccagtattatatgctgaattcagagattaagcca agcttgagtattctatagttcacctaaat 3’
3’ acattatgctgagtgatatcccgcttaa gggacggtcataatatacgacttaagtctctaattcggttgga actcataagatatcaagtggattta 5’
+ Ligase and ATP
Ligation with ligase and ATP
5‘aattccctgccagtattatatgctgaattcagagattaagcca
3‘
3‘
gggacggtcataatatacgacttaagtctctaattcggttgga 5‘
+
5’ tgtaatacgactcactatagggcg
3’ acattatgctgagtgatatcccgcttaa
agcttgagtattctatagttcacctaaat 3’
actcataagatatcaagtggattta 5’
5’ tgtaatacgactcactatagggcg aattccctgccagtattatatgctgaattcagagattaagcca agcttgagtattctatagttcacctaaat 3’
3’ acattatgctgagtgatatcccgcttaa gggacggtcataatatacgacttaagtctctaattcggttgga actcataagatatcaagtggattta 5’
After ligation, the plasmid and the fragment it contains are a “recombinant DNA”
5’ tgtaatacgactcactatagggcgaattccctgccagtattatatgctgaattcagagattaagccaagcttgagtattctatagttcacctaaat 3’
3’ acattatgctgagtgatatcccgcttaagggacggtcataatatacgacttaagtctctaattcggttggaactcataagatatcaagtggattta 5’
lactose operon
This plasmid do alpha complementation, it allows what people call white and blue screening.
This plamid is a bit strange, why in the world would we insert the DNA fragment in the midle of
something called lacZ ?
Inserting something over there would disturb the lacZ sequence and inactivate it.
lacZ code for a protein, the alpha peptide which is necessary for ß-galactosidase to work..
This plasmid is used to transform cells lacking the lacZ sequence.
When the plasmide is inside such a bacterium it will “complement” it with this lacZ sequence.
The plasmid affords the sequenceto the bacterium: it complement it
The gene Z code for ß-galactosidase.
The sequence of this gene can be cut in two parts.
The proteins coded by the two parts (alpha and beta) should be present for ß-galactosidase
activity. The sum of the two polypeptides have the same activiy as the whole gene product.
Some mutated bacteria lack the alpha part: they don’t show any ß-galactosidase activity.
When these bacterias are grown with X-gal, their colony stay white.
When they are transformed by a plasmid containing the lac Z gene (coding the alpha part).
The plasmid afford the missing part; the clonies become blue when x-gal is in the culture
medium.
If a DNA fragment is inserted in the plasmid, it interrupts lacZ, and the colonies stay white.
A cell without plasmid would die in the presence of ampicillin, because it need
a gene to detroy the ampicillin in the medium.
The plasmid provide this gene.
If the plamid has no insert, the bacterium would produce an efficient ß-galactosidase,
And so, be able to hydrolyse X-Gal: the colony will be blue
If the plasmid has an insert, the alpha peptide will be absent, and, in presence of X-gal,
the colonies would remain white.
The the white colonies have a plamid with an insert.
mammalian plasmids
Some plasmids are modified so that they can replicate or transcribe in other hosts:
Bacterial plasmids can be engineered to be transfected in mammalian cells.
In bacterias, genes are polycistronic.
In mammalian cells, genes are mostly monocistronic.
The promoter should be a good mammalian promoter ( the promoter of a mammalian viruses,
like Cytomegalo virus or SV40 virus) .
An intron is also added.
To construct the poly A tail of the mRNA a polyadenylation signal is added.
The cDNA to be expressed is inserted between the intron and the polyadenylation signal.
Transduction is the process by which genetic material,
e.g. DNA or siRNA, is inserted into a cell.
Common techniques in molecular biology are the use of
viral vectors (including bacteriophages), electroporation,
or chemical reagents that increase cell permeability.
Transfection and transformation are more common terms,
although these sometimes imply expression of the genetic
material as well.
Akt = PKB
Akt-P = Ser-473-phosphorylated Ak
p27: This gene encodes a cyclin-dependent kinase inhibitor
Figure 2. Expression levels of various proteins following PTEN transduction
into glioblastoma cell line A172. PTEN is highly expressed after transduction.
Total Akt protein expression has not changed following PTEN transduction,
whereas levels of actived Akt (Ser-473-phosphorylated Akt) are reduced after
PTEN transduction. Conversely, p27 protein accumulates after PTEN transduction.`
ß-actin expression remains constant, showing that equal amounts of protein were
loaded in each well
INTERNATIONAL JOURNAL OF ONCOLOGY 21: 1141-1150, 2002
Genetic profile, PTEN mutation and therapeutic role of PTEN in glioblastomas XING FAN,
YAN AALTO, STEPHEN G. SANKO, SAKARI KNUUTILA, DAVID KLATZMANN andJAVIER S.
CASTRESANA
GFP
The phMGFP Vector contains the open reading frame for the Monster Green Fluorescent Protein
cloned into a mammalian expression vector. The MGFP is encoded by an improved synthetic version
of thegreen fluorescent protein gene originally cloned from Montastrea cavernosa(Great Star Coral).
CMV Enhancer/Promoter: This CMV enhancer/promoter allows strong, constitutive expression.
Chimeric Intron: is composed of the
5´-donor site from the first intron of the human β-globin and the branch and
3´- acceptor site from the intron between the leader and body of an immunoglobin gene
heavy chain variable region. Transfection studies have demonstrated that the presence of
an intron flanking the cDNA insert frequently increases the level of gene expression
T7 Promoter: A T7 RNA polymerase promoter is located downstream of the chimeric intron
Green fluorescent protein (GFP) is commonly used to monitor gene expression and
protein trafficking within intact cells. GFP fusion proteins are easily visualized by
standard fluorescence microscopy to track realtime subcellular localization of a
protein of interest.
Luc
The pGL2 Luciferase Reporter Vectors(a) provide a basis for the quantitative analysis
of factors that potentially regulate mammalian gene expression. These factors may
be cis-acting, such as promoters and enhancers, or trans-acting, such as various
DNA-binding factors. The pGL2 Vectors carry the coding region for firefly ( Photinus
pyralis) luciferase, used to monitor transcriptional activity in transfected eukaryotic
cells.
CAT
The pCAT®3-Basic Vector lacks eukaryotic promoter and enhancer sequences,
allowing maximum flexibility in cloning putative regulatory sequences.
Expression of CAT activity in cells transfected with this plasmid depends on insertion
and proper orientation of a functional promoter upstream from the intron and the CAT gene.
Potential enhancer elements can also be inserted upstream of the promoter or in the BamH I or Sal I sites
downstream of the CAT transcription unit.
trypanosomal plasmids
plasmids of E. histolytica
plant plasmids and GMO
Large gall formed at the base of the stem
of a rose bush
Agrobacterium causes Crown gall disease.
This disease occurs when the soil bacterium Agrobacterium tumefaciens (A. tumefaciens )
enters a stem through a wound site (crown is the stem just above the soil surface).
This causes proliferation of tissue, like cancer growth (gall) and can occur on many
dicot species (grape, fruit and nut trees etc.).
Basically, the bacterium transfers part of its DNA to the plant, and this DNA integrates into
the plant’s genome, causing the production of tumours and associated changes in plant metabolism.
http://helios.bto.ed.ac.uk/bto/microbes/crown.htm
The unique mode of action of A. tumefaciens has enabled this bacterium to be used as
a tool in plant breeding. Any desired genes, such as insecticidal toxin genes or herbicide-resistance genes,
can be engineered into a bacterial plasmid, the Ti plasmid and thereby inserted into the plant genome.
Most of the genes involved in crown gall disease are not borne on the chromosome of A. tumefaciens
but on a large plasmid, termed the Ti (tumour-inducing) plasmid. In the same way, most of the genes
that enable Rhizobium strains to produce nitrogen-fixing nodules are contained on a large plasmid
termed the Sym (symbiotic) plasmid. Thus, the characteristic biology of these two bacteria is a
function mainly of their plasmids, not of the bacterial chromosome.
In natural conditions, the motile cells of A. tumefaciens are attracted to wound sites by chemotaxis
(acetosyringone 10-7 Molar). Acetosyringone plays a further role in the infection process, because at higher
concentrations (about 10-5 to 10-4 Molar) than those that cause chemotaxis it activates the virulence
genes (Vir genes) on the Ti plasmid. These Vir genes coordinate the infection process; they:
1. lead to the production of proteins (permeases) that are inserted in the bacterial
cell membrane for uptake of nutritive compounds (opines) that will be produced
by the tumours
2. cause the production of an endonuclease - a restriction enzyme - that excises part
of the Ti plasmid termed the T-DNA (transferred DNA).
When integrated into the plant genome, the genes on the T-DNA code for:
* production of cytokinins
* production of indoleacetic acid
* synthesis and release of novel plant metabolites - the opines and agrocinopines.
The plant hormones upset the normal balance of cell growth, leading to the production of galls
and thus to a nutrient-rich environment for the bacteria. The opines are unique amino acid derivatives,
different from normal plant products, and the agrocinopines similarly are unique phosphorylated
sugar derivatives. All these compounds can be used by the bacterium as the sole carbon and energy source,
and because they are absent from normal plants they provide Agrobacterium with a unique food source
that other bacteria cannot use.
Genetic engineering of plants with A. tumefacien
The transgenic tomatoes do not express the gene for polygalacturonase, an enzyme that degrades pectin,
leading to softening of the fruit tissues. As a result, the tomatoes can be left on the plant for longer
to accumulate flavour components and they also give a better consistency of tomato pastes.
Several crop plants have been engineered to express the insecticidal toxin gene of Bacillus thuringiensis
(see Profile on Bacillus thuringiensis), so that insects attempting to eat these plants are killed.
This is highly successful, but has the potential disadvantage that continuous exposure of insects to
the toxin will select for the development of toxin resistance. Cropping strategies must be devised
to "manage" this.
Several crops also have been engineered for resistance to herbicides such as glyphosate, so that the herbicide can be used
Other transgenic strategies being explored or commercialised include:
Engineering for virus resistance by incorporation of viral coat protein genes or antisense RNA
Engineering for resistance to fungal pathogens, by enhanced expression of fungal wall-degrading enzymes (chitinase and gl
Engineering of plants so that, during a late stage in the development of their seeds, they express a gene that renders the see
gene knockout
A gene knockout is a genetically engineered organism that carries one or more genes
in its chromosomes that has been made inoperative. So far such organisms have been engineered
chiefly for research purposes.
Knockout is accomplished through a combination of techniques, beginning in the test tube with
a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture
Individual cells are genetically transformed with the construct and--for knockouts in multi-cellular
organisms--ultimately fused with a stem cell from a nascent embryo.
The construct is engineered to recombine with the target gene, which is accomplished by
incorporating sequences from the gene itself into the construct. Recombination then occurs
in the region of that sequence within the gene, resulting in the insertion of a foreign sequence
to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated
into a nonfunctional protein, if it is translated at all.
homologous recombination: Suppose that we wanted to replace
the sequence in red by nother one.
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
tatctggttg
agtataaaga
tggttagtaa
atttgtatta
gagttaggat
ttctgatcta
gaattggggt
agcaggcgcg
agttgagtaa
ttggagggca
agttgctgtg
ctttanntaa
gtaattgagt
tgaatattcg
ttaaaaggaa
tataagatgc
gttaggggat
aggattggat
atcaatacta
gtatggtcac
tgcggcttaa
cagattaaga
gagtgatttg
tgcctataag
acatttcaat
gtgggaaaaa
tgggccgcac
tattaggcta
ggaaaactca
aggaattcct
acaccgcccg
agatagaaaa
ccgtaggtga
atcctgccag
ccaagtagga
agtacaagga
gtacaaagtg
gccacgacaa
tcaatcagtt
tcgacatcgg
taaattaccc
aatcaattct
agtctggtgc
attaaaacgc
gtgaagtttc
tgttattact
agcatgggac
caattggggt
acgagagcga
cgaagacgat
gaaattcaga
ccttgttcag
aaggctgaaa
tttgactcaa
gttctttcat
tcaggttaat
acagaaatgt
tgtcctattt
gaaaaaggaa
gcgcgctaca
tgtctaatag
aaagaacgta
tgtaatatcg
tcgctcctac
atggatttaa
acctgcggaa
tattatatgc
tgaaactgcg
tagctttgtg
gccaatttat
ttgtagaaca
ggtagtatcg
agagggagct
actttcgaat
tgaaggaatg
cagcagccgc
tcgtagttga
tagaaatgtt
ttgaataaaa
aatgctgagg
gattcagaaa
aagcatttca
cagataccgt
tgtacaaaga
aacttaaaga
cttaaaggaa
cacgggaaaa
gatttattgg
tccggtaacg
tcgcaagaac
taattgttag
gcattcagca
atggagttac
gtagggatag
catgacaggg
agtcattaac
cgattgaata
atctccttat
ggatcatta
tgatgttaga
gacggctcat
aatgataaag
gtaagtaaat
cacagtgttt
aggactacca
ttacagatgg
tgaagaggta
agtaggaggt
ggtaattcca
attaaaatgt
aaattaaaat
taaggtgttt
ggatgtcaat
ataacgggag
ctcaactggg
cgtagtccta
tgaagaaaca
gaaatcttga
ttgacggaag
cttaccaaga
gtagtggtgc
aacgagactg
aggtgcgtaa
ttatctaatt
ataacaggtc
tagagagcat
taagtggtgt
ataaatgatt
tcgagatgaa
aagaggtgaa
ttagaggaag
gattaagcca
tataacagta
ataatacttg
tgagaaatga
aacaagtaac
agattataac
ctaccacttc
gtgacgacac
aaattctcct
gctccaatag
gattttatac
caaagaagga
aaagcaaaac
tagacatttc
aggtgaaaat
tccattaatc
actataaacg
ttgtttctaa
gtttatggac
ggcacaccag
ccgaacagta
atggccgttc
aaacctatta
gtaccacttc
tcgattagaa
tgtgatgccc
tttatcattt
accgagattg
ggaattattt
tacgtccctg
attctaggat
gagaagtcgt
tgcatgtgta
atagtttctt
agacgatcca
cattctaagt
caatgagaat
ggataacgag
taaggaaggc
ataactctag
acgaaatcaa
tgtatattaa
attttgaaga
gacnnttcaa
attatgttaa
gagagaagga
ccatgatcgg
aagaacgaaa
atgtcaacca
atccaagtat
ttcaggggga
gagtggagcc
gaaggaatga
ttagttggtg
attagttttc
ttaaagggac
ctcttttaac
ttagacatct
acaccttatt
aaatagttaa
gttttgaacg
ccctttgtac
tctgtcttat
aacaaggttt
Regardless of what you want to insert, you must include some flanking DNA that is identical
in sequence to the targeted locus. Lets color them in blue and green respectively. Let’s represent
sequence in the next drawings by a blue, a red and a green line.
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
tatctggttg
agtataaaga
tggttagtaa
atttgtatta
gagttaggat
ttctgatcta
gaattggggt
agcaggcgcg
agttgagtaa
ttggagggca
agttgctgtg
ctttanntaa
gtaattgagt
tgaatattcg
ttaaaaggaa
tataagatgc
gttaggggat
aggattggat
atcaatacta
gtatggtcac
tgcggcttaa
cagattaaga
gagtgatttg
tgcctataag
acatttcaat
gtgggaaaaa
tgggccgcac
tattaggcta
ggaaaactca
aggaattcct
acaccgcccg
agatagaaaa
ccgtaggtga
atcctgccag
ccaagtagga
agtacaagga
gtacaaagtg
gccacgacaa
tcaatcagtt
tcgacatcgg
taaattaccc
aatcaattct
agtctggtgc
attaaaacgc
gtgaagtttc
tgttattact
agcatgggac
caattggggt
acgagagcga
cgaagacgat
gaaattcaga
ccttgttcag
aaggctgaaa
tttgactcaa
gttctttcat
tcaggttaat
acagaaatgt
tgtcctattt
gaaaaaggaa
gcgcgctaca
tgtctaatag
aaagaacgta
tgtaatatcg
tcgctcctac
atggatttaa
acctgcggaa
tattatatgc
tgaaactgcg
tagctttgtg
gccaatttat
ttgtagaaca
ggtagtatcg
agagggagct
actttcgaat
tgaaggaatg
cagcagccgc
tcgtagttga
tagaaatgtt
ttgaataaaa
aatgctgagg
gattcagaaa
aagcatttca
cagataccgt
tgtacaaaga
aacttaaaga
cttaaaggaa
cacgggaaaa
gatttattgg
tccggtaacg
tcgcaagaac
taattgttag
gcattcagca
atggagttac
gtagggatag
catgacaggg
agtcattaac
cgattgaata
atctccttat
ggatcatta
tgatgttaga
gacggctcat
aatgataaag
gtaagtaaat
cacagtgttt
aggactacca
ttacagatgg
tgaagaggta
agtaggaggt
ggtaattcca
attaaaatgt
aaattaaaat
taaggtgttt
ggatgtcaat
ataacgggag
ctcaactggg
cgtagtccta
tgaagaaaca
gaaatcttga
ttgacggaag
cttaccaaga
gtagtggtgc
aacgagactg
aggtgcgtaa
ttatctaatt
ataacaggtc
tagagagcat
taagtggtgt
ataaatgatt
tcgagatgaa
aagaggtgaa
ttagaggaag
gattaagcca
tataacagta
ataatacttg
tgagaaatga
aacaagtaac
agattataac
ctaccacttc
gtgacgacac
aaattctcct
gctccaatag
gattttatac
caaagaagga
aaagcaaaac
tagacatttc
aggtgaaaat
tccattaatc
actataaacg
ttgtttctaa
gtttatggac
ggcacaccag
ccgaacagta
atggccgttc
aaacctatta
gtaccacttc
tcgattagaa
tgtgatgccc
tttatcattt
accgagattg
ggaattattt
tacgtccctg
attctaggat
gagaagtcgt
tgcatgtgta
atagtttctt
agacgatcca
cattctaagt
caatgagaat
ggataacgag
taaggaaggc
ataactctag
acgaaatcaa
tgtatattaa
attttgaaga
gacnnttcaa
attatgttaa
gagagaagga
ccatgatcgg
aagaacgaaa
atgtcaacca
atccaagtat
ttcaggggga
gagtggagcc
gaaggaatga
ttagttggtg
attagttttc
ttaaagggac
ctcttttaac
ttagacatct
acaccttatt
aaatagttaa
gttttgaacg
ccctttgtac
tctgtcttat
aacaaggttt
The next step is to design and fabricate the DNA construct you want to insert in place of the red sequence.
This construct may contain any DNA sequence, but must include some flanking DNA (blue and green).
In addition to the positive selection marker (e.g. antibiotic resistance) often a negative selection marker
(e.g. thymidine kinase, tk) is added to the replacement vector.
The negative marker is outside the region of sequence similarity between the vector and the targeted locus.
The positive selection marker, neo, code for a protein that inactivates G418, a neomycine homolog.
The negative marker code for thymidine kinase (tk). If tk is incorpotated, this is due to a non-homologous
recombination event. In this case, tk kill the cell by modifying the molecules of gancyclovir added
to the medium
So the cell in which happen the right recomnination will stay alive in a medium containing Neomycin and
gancyclovir.
During meiosis and mitosis when homolgous chromosomes align along the metaphase plane,
the engineered construct finds the targeted gene and recombination takes place within the homolgous
(meaning identical in this case) sequences The recombination may take place anywhere within the
flanking DNA sequences and the exact location is determined by the cells and not the investigators.
Once the cells have performed their part of the procedure,
the end result is a new piece of DNA inserted into the chromosome. The rest of the genome is unaltered
but the single targeted locus has been replaced with the engineered construct and some of its flanking
DNA.
The original engineered construct has taken up the targeted gene of interest but since it cannot replicate
in a nucleus, it is lost quickly in dividing cells while the modified chromosome replicates faithfully,
including its new insert.
Cells that have undergone homologous recombination can be selected by addition of antibiotic
to the growth medium (positive selection). Notice that the negative selection marker is not incorporated
into the chromosome by homologous recombination.
Look now at the green sequences which were addded on the drawing. This sequence recognize
the long and the short fragments:
Bam HI -Bam HI 5.5 kb and Bam HI -Bam HI 15 kb
We not only synthetize this fragment, but in addition we labelled it with 32P or 33P, both are
radioactive isotopes of phosphorous (It’s time for you to study how we do that!)
Then, we prepare and purify the DNA of this organism. If the organism is a mouth, we
prepare genomic DNA from a tail fragment.
We hydrolyse completely this DNA with Bam HI to be sure to get the two fragments:
Bam HI -Bam HI 5.5 kb and Bam HI -Bam HI 15 kb
We submit the hydrolysate to the Southern Analysis.
after an electrophoresis on agarose gel and denaturation (separation of the two strands),
the gel is put on a device which allow the tranfer of the DNA on a nylon filter. The denatured
DNA is attached covalently to the membrane by a short UV irradiation(both strand are still
separated).
The filter is put in a freezer bag. The (green) labelled probe is added.
The probe binds to its complement either the 15 or the 4.5 fragment