Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5992_Библиотеки_им_академика_М_И_Перельмана.pdf
Genetic Recombination
195
develops into floral tillers. Around 0.3 mL of DNA solution is injected at a point above tiller node
until several drops of solution come out from top of young inflorescence.
Advantages
1. Instrument is simple and cheap.
2. This technique does not require protoplast.
3. The method may prove useful for gene transfer into cereals, which do not regenerate from
cultured cell easily.
4. It is a simple technology to handle.
Limitations
1. It is less specific.
2. The frequency of transformation is very low.
3. It is less efficient.
Biolistics or microprojectiles for DNA transfer
Biolistic or particle bombardment is a physical technique that uses accelerated microprojectiles to
deliver DNA or other molecules into intact tissues and cells. The method was developed initially to
transfer genes into plants by Sanford. The technique of biolistics transformation is relatively new,
and it is a novel method amongst the physical methods for artificial transfer of exogenous DNA.
This method is better in efficiency and does not need protoplast. This technique can be used for any
plant cells, root sections, seed embryos and pollen grains. The gene gun is a device that literally
fires DNA into target cells. The DNA to be transformed into the cells is coated on to microscopic
beads made up of either gold or tungsten. The beads are carefully coated with DNA and are then
attached to the end of the plastic bullet and loaded into the firing chamber of the gene gun. An
explosive force fires the bullet down the barrel of the gun towards the target cells that lie just
beyond the end of the barrel. When the bullet reaches the end of the barrel, it is caught and stopped,
but the DNA-coated beads continue towards the target cells. Some of the beads pass through the
cell wall into the cytoplasm of the target cells. Here, the bead and the DNA dissociate and the cells
become transformed. Once inside the target cells, the DNA is solubilized and may be expressed.
Applications
1. Genomes of subcellular organelles have been accessible to genetic manipulation by biolistic
method.
2. It is used successfully to transform cotton, soybean, spruce, sugarcane, corn, papaya, sunflower,
maize, rice, tobacco, wheat, etc.
3. Method can be applied to filamentous fungi and yeast.
4. Mitochondria of plants and chloroplast of Chlamydomonas are transformed.
5. The particle gun is used with pollen, early stage embryoids, meristems and somatic embryos.
Advantages
1. Walled intact cells can be penetrated.
Textbook of Pharmaceutical Biotechnology
196
2. Requirement of protoplast is avoided.
3. Manipulation of genome of subcellular organelles can be achieved.
Liposome-mediated gene transfer
Introduction of DNA into cell via liposomes is known as lipoinfection. Liposomes are small lipid
vesicles, in which large numbers of plasmids are enclosed. They can be induced to fuse with
protoplast vesicle device, such as PEG. Therefore, they are used for gene transfer. This method
can be used for both transient and stable transfection. It can be used for adherent cells, primary
cell lines, and suspension cultures (Fig 13.3). This method can also be used in the transfer for the
production of transgenic animal.
Some other techniques used for liposomes
For the following protocol, Lipofectamine reagent from Invitrogen Corporation is used.
Lipofectamine reagent is a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-
N-[2(sperminecardox-ido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and
neutral lipid dioleoylphosphatidylethanolamine (DOPE) in membrane-filtered water (Catalogue
Number 18324; Invitrogen Corporation, Carlsbad, California, US) (Hawley-Nelson, 1993; Shih,
1997).
1. Around 40,000 cells per well of a 24-well plate are placed in 0.5 mL of the appropriate
complete growth medium (10% serum is added, if needed).
2. The cells are incubated at 37°C in a CO
2
incubator until the cells are 50–80% confluent. It
takes around 20 h.
elease of plasmid
Protoplast
usion of liposomes in the protoplast
Liposomes
containing plasmid
Figure 13.3 Liposome-mediated gene transfer.
Genetic Recombination
197
3. About 3 g DNA is diluted into 25 L medium without serum for each well and mixed
thoroughly.
4. About 3 L Lipofectamine reagent is diluted with 25 L medium without serum for each well
and mixed thoroughly.
5. The diluted DNA (step 3) and Lipofectamine reagent (step 4) are combined together and
incubated at room temperature for 30 min. During incubation, the DNA–liposome complexes
are formed.
6. The medium in the cells is replaced with 0.2 mL transfection medium without serum.
7. About 0.15 mL medium without serum is added to the tube containing the complexes for each
well.
8. The cells are then incubated with the complexes for about 10 h at 37°C in a CO
2
incubator.
The incubating time shall be flexible and depends on used cell type.
9. About 0.4 mL growth medium containing double the 2× normal concentration of the serum is
added without removing the transfection mixture.
10. Later, the medium is replaced with fresh and complete medium.
11. The cell extracts for transient gene expression are assayed at 24–72 h after transfection,
depending on the cell type and promoter activity.
12. To obtain stable transfectants, the cells are passaged 1:10 into the selective medium after 72 h of
transfection for the reporter gene transfected.
For use as gene transfer vehicles, liposomes are prepared by adding an appropriate mix of bilayer
constituents to an aqueous solution of DNA molecules. In this aqueous environment, phospholipid
hydrophilic heads associate with water, whereas hydrophobic tails self-associate to exclude water
from within the lipid bilayer. This self-organizing process creates discrete spheres of continuous
lipid bilayer membrane enveloping a small quantity of DNA solution. The liposomes are then ready
to be added to target cells. Germline transgenesis is possible with liposome-mediated gene transfer
and ES cells have been successfully transfected by liposomes.
Advantages
1. Long-term stability
2. Protection of nucleic acid from degradation
3. Low toxicity
4. Simplicity of technique
Chemical Methods
PEG-mediated transfection
PEG stimulates endocytosis, and therefore, DNA uptake occurs. This method is utilized only for
protoplast. PEG-based vehicles are less toxic and more resistant to nonspecific protein adsorption
making them attractive for nonviral gene delivery.
The protoplasts are kept in solution containing PEG. The molecular weight of PEG used is
8000 Da with final concentration of 15%. Calcium chloride is added to it along with sucrose and
glucose that act as osmotic buffering agents. To reduce the effects of nuclease present, the carrier
Textbook of Pharmaceutical Biotechnology
198
DNA from salmon or herring sperm may also be added. After exposure of the protoplast to exogenous
DNA in presence of PEG and other chemicals, PEG is allowed to be removed. Intact surviving
protoplasts are then cultured to form cells with walls and colonies in turn. After several passages in
selectable medium, frequency of transformation is calculated. PEG-PBLG nanoparticle-mediated
HSV-TK/GCV gene therapy is also reported for oral squamous cell carcinoma.
Calcium phosphate-mediated DNA transfer
The process of transfection involves the admixture of isolated DNA (10–100 mg) with solution of
calcium chloride and potassium phosphate under conditions that permit precipitation of calcium
phosphate. The technique is used for introducing DNA into mammalian cells. The cells are then
incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells shall
take the calcium phosphate DNA precipitate by endocytosis. The transfection efficiencies using
calcium phosphate can be quite low (in the range of 1–2%). It can be increased, if very high purity
DNA is used, and the precipitate is allowed to form slowly. The technique has been developed
whereby cell taking up exogenous DNA could be increased to 20%.
Limitations
1. Integrated genes undergo substantial modification.
2. Integration with host cell chromosome is random.
3. Many cells do not like solid precipitate adhering to them and the surface of their culture vessel.
4. Frequency is very low.
Due to the above limitations, transfection applied to somatic gene therapy is limited. The calcium
phosphate method of DNA transfer is reproducible and efficient, but there is a narrow range of
optimum conditions.
Transfer of DNA by polycation-DMSO
For DNA transfer by polycation, polybrene is used to increase the adsorption of DNA to the cell
surface followed, by a brief treatment with 25–30% DMSO to increase membrane permeability and
enhance uptake of DNA. In this technique, carrier DNA is not required and stable transformants are
produced. This method works with mouse fibroblast and chick embryo.
DNA transfer by DEAE-D
The DNA can also be transferred with the help of DEAE-dextran. DEAE-dextran may be used in
the transfection medium containing DNA. This is polycationic, high molecular weight substance,
convenient for transient assays in cos cells. When diethylaminoethyl-dextran treatment is coupled
with DMSO, up to 80% transformed cells can express the transferred gene. It is established that
serum inhibits this transfection. Therefore, cells are washed nicely to make them serum free. It does
not appear to be efficient for the production of stable transfectants. The treatment with chloroquinine
increases transient expression of DNA.
Advantages
1. It is a simple method.
Genetic Recombination
199
2. It can be used for transient cells, which can not survive even short exposure to calcium
phosphate.
3. It is economically viable and cheep technique.
Different steps involved in these techniques are as follows:
1. The growing cells are harvested exponentially by trypsinization and transferred into 60-mm
tissue culture dishes at a density of 105 cells per dish.
2. About 5 mL complete growth medium is added to it.
3. It is then incubated for 24 h at 37°C with 5% CO
2
.
4. DNA/DEAE-dextran/TBS-D (Tris-buffered saline with dextrose) solution is prepared by
mixing 2 mg of supercoiled plasmid DNA into 1 g/mL DEAE-dextran in TBS-D.
5. The medium is removed and washed three times with phosphate buffered saline (PBS) and
twice with TBS-D. DNA/DEAE- dextran/TBS-D solution (250 L) is added to it.
6. It is then incubated at 37°C for 60 min with 5% CO
2
.
7. DNA/DEAE-dextran/TBS-D solution is removed and washed with TBS-D thrice and twice
with PBS.
8. About 5 mL of medium supplemented with serum and chloroquine (0.1 mM) is added
to it.
9. It is incubated for 4 h at 37°C with 5% CO
2
.
10. The medium is removed and washed with serum-free medium three times.
11. About 5 mL of medium supplemented with serum is added to it and incubated at 37°C for
48 h with 5% CO
2
.
12. The cells are harvested after 48 h of incubation.
13. The RNA or DNA is analysed by hybridization or the expressed protein is analysed by
radioimmunoassay, immunoblotting or immunoprecipitation.
Use of polybrene
Several polycations, including polybrene (1,5-dimethyl-1,5-diazaundecamethylene poly-methobromide)
(Chaney, 1986) and poly-L-ornithine (Nead, 1995) have been used in gene transfection with the DMSO
enhancement.
The steps involved are as follows:
1. The cells are harvested exponential by trypsinzationin and replanted at a density of 5000 cells/
mm
2
in 10 mL MEM_ containing 10% fetal calf serum.
2. It is incubated at 37°C 24 h in 5% CO
2
.
3. The medium is replaced with 3 mL prewarmed medium containing serum, 10 g DNA and
30 g polybrene (37°C). The medium is mixed thoroughly before adding polybrene.
4. It is the then incubated for 12 h with agent and shaken each hour.
5. The medium is removed and to it added 5 mL of 30% DMSO in serum-containing
medium.
6. After 4 min of incubation, the DMSO solution is aspirated, and the cells are washed twice
with warm (37°C) serum-free medium. A total of 10 mL of complete medium containing 10%
fetal calf serum is added to it.
Textbook of Pharmaceutical Biotechnology
200
Gene transfer through peptide
A series of peptide sequences are able to bind to, and condense, DNA to make it more amenable for
entry into cells. For example, the tetrapeptide ‘serine-proline-lysine-lysine’ (present on the
C terminal of the histone H1 protein) helps in DNA transfer. Lysine is a positively charged amino acid.
Its side chains help to counteract the negatively charged phosphate DNA backbone and allow the
DNA molecules to pack closely to each other. Tyrosine-lysine-alanine-(lysine)
8
-tryptophan-lysine is
a peptide that is very effective to form complexes with DNA. DNA-binding peptides that can be
coupled to cell-specific ligands can also be synthesized. It allows receptor-mediated targeting of the
peptide/DNA complexes to specific cell types. Rational design of peptide sequences has been used
to develop synthetic DNA-binding peptide.
HORIZONTAL GENE TRANSFER TECHNOLOGY
Horizontal gene transfer (HGT) is a process in which a prokaryotic cell can acquire genes from other
microbes of the same generation, which, in some cases, can be a different species, or even a different
genus than the donor. The technology of HGT was first described in Japan in 1959. The related publication
has demonstrated the transfer of antibiotic resistance between different species of bacteria.
There are three types of HGT as follows:
1. Transformation
2. Transduction (bacterial)
3. Conjugation (bacterial)
Transformation
In molecular biology, transformation is the genetic alteration of a cell resulting from the direct
uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its
surrounding and taken up through the cell membrane(s). Transformation occurs most commonly in
bacteria. It can also be affected by artificial means. Bacteria that are capable of being transformed,
whether naturally or artificially, are called competent. Transformation is one of three processes
by which exogenous genetic material may be introduced into bacterial cell, the other two being
conjugation (transfer of genetic material between two bacterial cells in direct contact) and
transduction (injection of foreign DNA by a bacteriophage into the host). Transformation is also
used to describe the insertion of new genetic material into nonbacterial cells, including animal
and plant cells. However, because transformation has a special meaning in relation to animal cells
indicating progression to a cancerous state, the term should be avoided for animal cells while
describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic
cells is usually called transfection.
Mechanism of Transformation
1. Bacteria: The bacterial transformation may be referred to as a stable genetic change brought
about by the uptake of naked DNA (DNA without associated cells or proteins). Competence
Genetic Recombination
201
refers to the state of being able to take up exogenous DNA from the environment. Two forms
of competence exist: natural and artificial.
2. Natural competence: About 1% of bacterial species are capable of naturally taking up DNA
under laboratory conditions. Many more are able to take it up in their natural environments.
Such bacteria carry sets of genes that provide the protein machinery to bring DNA across the
cell membrane(s).
3. Artifi cial competence: The artificial competence is induced by laboratory procedures. It
involves making the cell passively permeable to DNA by exposing it to conditions that do
not normally occur in nature. The efficiency with which a competent culture can take up
exogenous DNA and express its genes is known as transformation effi ciency.
The calcium chloride transformation is an ideal technique for this purpose. The chilling
cells in the presence of divalent cations such as Ca
2+
(in CaCl
2
) prepare the cell membrane to
become permeable to plasmid DNA. The cells are incubated on ice with the DNA and then
briefly heat shocked (at 42°C for 30–120 s) allowing the DNA to enter the cells. This method
works very well for circular plasmid DNA. An excellent preparation of competent cells
gives ~10
8
colonies per microgram of plasmid. The noncommercial and good preparations
give around 10
5
–10
6
transformants per microgram of plasmid. Interestingly, the cells that
are naturally competent are usually transformed more efficiently with linear DNA than with
plasmid DNA.
4. Plasmid transformation: The transformation usually produces a mixture of relatively few
transformed cells and an abundance of nontransformed cells. A method is therefore needed to
identify the cells that have acquired the plasmid. In order to be stably maintained in the cell,
a plasmid DNA molecule must contain an origin of replication. This allows it to be replicated
in the cell independent of the replication of the cell’s own chromosome. The method usually
consists of using a plasmid that contains a gene that gives the bacterial cells resistance to an
antibiotic that they are naturally sensitive to. The mixtures of cells are then plated on medium
that contains the antibiotic. Only the transformed cells are able to grow, and the cells that do
not take up the plasmid are killed in the medium.
5. Induction in plants: A number of mechanisms are available to transfer DNA into plant cells.
a. Agrobacterium-mediated transformation is the easiest and most simple plant transformation.
Plant tissue (often leaves) are cut into small pieces (10 u 10 mm) and soaked for 10 min in
a fluid containing suspended Agrobacterium. Some cells along the cut will be transformed
by the bacterium that inserts its DNA into the cell. It is then placed on selectable rooting
and shooting media to allow plants to regrow. Some plant species can be transformed just
by dipping the flowers into suspension of Agrobacterium.
b. Particle combardment: This method also allows transformation of plant plastids. The
transformation efficiency is lower than in Agrobacterium-mediated transformation, but
most plants can be transformed with this method. The particles of gold or tungsten are
coated with DNA and then shot into young plant cells or plant embryos. Some genetic
materials shall stay in the cells and transform them.
c. Electroporation: The transient holes in cell membranes are made using electric shock.
This treatment allows DNA to enter as described above for bacteria.
Textbook of Pharmaceutical Biotechnology
202
Transduction
The technique of transduction was discovered by Norton Zinder and Joshua Lederberg at
the University of Wisconsin, Madison in 1951. This is a common tool used by molecular
biologists to stably introduce a foreign gene into a host cell’s genome. In this process, DNA
is transferred from one bacterium to another by a virus. It is also the process whereby foreign
DNA is introduced into another cell via a viral vector. The desired genetic material is packed
into a suitable plant virus, and this modified virus is allowed to infect the plant. If the genetic
material is DNA, it can recombine with the chromosomes to produce transformant cells.
However, genomes of plant viruses usually consist of single-stranded RNA that replicates in
the cytoplasm of the infected cell. For such genomes, this technique is a form of transfection
and not a real transformation, because the inserted genes never reach the nucleus of the cell
and do not integrate into the host genome. The progeny of such infected plants is virus free and
also free of the inserted gene.
Generalized transduction may occur in two main ways, namely, recombination and headful
packaging. If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the
virus shall control the machinery of the cell for use in replicating its own viral DNA. If by chance,
bacterial chromosomal DNA is inserted into the viral capsid used to encapsulate the viral DNA, this
may lead to generalized transduction. If the virus replicates using headful packaging, it attempts to
fill the nucleocapsid with genetic material.
The new virus capsule now loaded with part bacterial DNA continues to infect another bacterial
cell. This bacterial material may become recombined into another bacterium upon infection.
When the new DNA is inserted into this recipient cell it can fall to one of the following three fates:
1. The DNA shall be absorbed by the cell and be recycled for spare parts.
2. If the DNA is originally a plasmid, it recircularizes inside the new cell and becomes a plasmid
again.
3. If the new DNA matches with a homologous region of the chromosome of recipient cell, it
shall exchange DNA material similar to the actions in conjugation.
Conjugation
The process of conjugation involves the transfer of genetic material between bacterial cells by direct
cell–cell contact or by a bridge-like connection between two cells. Discovered in 1946 by Joshua
Lederberg and Edward Tatum, conjugation is a mechanism of HGT as in case of transformation
and transduction; although these two other mechanisms do not involve cell–cell contact.
The bacterial conjugation is often incorrectly regarded as the bacterial equivalent of sexual
reproduction or mating, since it involves the exchange of genetic materials. During conjugation,
the donor cell provides a conjugative or mobilizable genetic element that is most often a plasmid
or transposon. Most conjugative plasmids have systems ensuring that the recipient cell does not
already contain a similar element.
Genetic Recombination
203
The genetic information transferred is often beneficial to the recipient. The benefits derived
are antibiotic resistance, and xenobiotics tolerance or the ability to use new metabolites. Such
beneficial plasmids may be considered bacterial endosymbionts.
Mechanism of Conjugation
The prototypical conjugative plasmid is the F-plasmid or F-factor. It is an episome with a length of
about 100 kb. An episome is a plasmid that can integrate itself into the bacterial chromosome by
homologous recombination. It carries its own origin of replication, the oriV and an origin of transfer
or oriT. There can only be one copy of the F- plasmid in a given bacterium, either free or integrated,
and bacterium that possesses a copy is called F-positive or F-plus (denoted F
+
). The cells that lack
of F- plasmids are called F-negative or F-minus (F
<
) and as such, can function as recipient cells.
The F- plasmid carries a tra and a trb locus, which together are about 33-kb long and consist
of about 40 genes. The tra locus includes the pilin gene and regulatory genes, which together form
pili on the cell surface. The locus also includes the genes for the proteins that attach themselves to
the surface of F
<
bacteria and initiate conjugation. The pili are not exactly the structures through
which DNA exchange occurs. Several proteins coded for the tra or trb locus seem to open a channel
between the bacteria and it is presumed that the traD enzyme, located at the base of the pilus,
initiates membrane fusion (Fig. 13.4).
Chromosomal DNA
-plasmid
Chromosomal DNA
Donor
ecipient
DNA polymerase
-plasmid-plasmid
Old donor
Ne donor
Figure 13.4 Conjugation of bacteria.
Textbook of Pharmaceutical Biotechnology
204
When conjugation is initiated by a signal, the relaxase enzyme creates a nick in one of the strands
of the conjugative plasmid at the oriT. The enzyme relaxase may work alone or in a combination
of over a dozen proteins known as relaxosome. In the F-plasmid system, the relaxase enzyme
is called TraI, and the relaxosome consists of TraI, TraY, TraM and the integration host factor
(IHF). The nicked strand or T-strand, is then unwound from the unbroken strand and transferred
to the recipient cell in a 5v-terminus to 3v-terminus direction. The remaining strand is replicated
either independent of conjugative action (vegetative replication beginning at the oriV) or in concert
with conjugation (conjugative replication similar to the rolling circle replication of lambda phage).
Conjugative replication may require a second nick before successful transfer can occur. It has been
recently claimed that conjugation is inhibited with chemicals that mimic an intermediate step of
this second nicking event.
Specialized Transduction
The specialized transduction occurs as a result of mistakes in the transition from a lysogenic to
lytic cycle of a virus. If a virus incorrectly removes itself from the bacterial chromosome, bacterial
DNA from either end of the phage DNA may be packaged into the viral capsid. The process of
specialized transduction leads to three possible outcomes as follows:
1. The bacterial DNA can match up with a homologous DNA in the recipient cell and exchange
it. The recipient cell thus has DNA from both itself and the other bacterial cell.
2. DNA can be absorbed and recycled for spare parts.
3. DNA can insert itself into the genome of the recipient cell as if still acting like a virus resulting
in a double copy of the bacterial genes.
HGT in Prokaryotes
The comparative analysis of bacterial, archaeal and eukaryotic genomes indicates that a significant
fraction of the genes in the prokaryotic genomes is subjected to horizontal transfer. The amount
and source of HGT sometimes may be linked to an organism’s lifestyle. For example, bacterial
hyperthermophiles exchange genes with archaea to a greater extent as compared to bacteria,
whereas, transfer of certain groups of eukaryotic genes is most common in parasitic and symbiotic
bacteria. Horizontal transfer events can be classified into distinct categories of (1) acquisition of
new genes, (2) acquisition of paralogs of existing genes and (3) xenologous gene displacement
whereby, a gene is displaced by a horizontally transferred ortholog from another lineage (xenolog).
All these types of HGT are common amongst prokaryotes, but their relative contributions differ in
different lineages. The horizontally transferred genes confer a selective advantage on the recipient
organism.
Several cases of acquisition of eukaryotic genes by bacteria seem to reveal the evolutionary
forces involved. For examples, isoleucyl-tRNA, whose acquisition from eukaryotes by several
bacteria is linked to antibiotic resistance, ATP/ADP translocases acquired by intracellular
parasitic bacteria, chlamydia and rickettsia and proteases that may be implicated in chlamydial
pathogenesis.