Animal biotechnology
lecture 2
Dr. Ziad Jaradat
1
Animal Biotechnology &
Transgenic Animals
• Since the early 1980s, fruit flies, fish, sea urchins, frogs,
laboratory mice and farm animals, such as cows, pigs,
and sheep have been successfully produced.
• The ability to manipulate the genome of the whole animal
and the production of transgenic animals has influenced
the science dramatically in the last 15 years.
• The procedure for introducing exogenous donor DNA into
a recipient cell is called Transfection.
• Chromosomes are taken up inefficiently so that intact
chromosomes rarely survived the procedure. Instead the
recipient cell usually get a part of the DNA.
2
• Now, with the advent of the recombinant
DNA, the possibility of introducing a
particular segment of DNA become possible.
However, still there are always some
problems of the stability of the new inserts
(transient transfectants).
• An exciting development of transfection
techniques is the application of DNA
technology to introduce genes into animals.
3
• An animal that gains new genetic information from the addition of
foreign DNA is described as Transgenic while the introduced DNA
is called the transgene.
• The transgenes are introduced into the pronuclei of fertilized eggs
by injection, and the injected embryos are incubated in vitro or
implanted into the uterus of a pseudopregnant female for
subsequent development.
What is Pronucleus? For a short time after fertilization, the male
pronucleus and female pronucleus exist separately.
• Female pronucleus; In the maturing of the ovum preparatory to
impregnation, a part of the germinal vesicle becomes converted
into a number of small vesicles, which aggregate themselves into a
single clear nucleus which travels towards the center of the egg and
is called the female pronucleus.
4
• Male pronucleus; In impregnation, the
spermatozon which enters the egg soon loses
its tail, while the head forms a nucleus, called
the male pronucleus, which gradually travels
towards the female pronucleus and eventually
fuses with it, forming the first segmentation
nucleus. The male pronucleus is larger than
the female’s and can be seen fairly easily
under a light microscope.
5
6
Synopsis of the transgenesis process;
• Plasmids carrying the gene of interest are injected into
the germinal vesicle (nucleus) of the oocyte or into the
pronucleus (before uniting with the gamete) of the
fertilized egg.
• The egg is implanted into a pseudopregnant mouse
• After birth, the recipient mouse can be examined to see
whether it has gained the foreign DNA and if so whether
it is expressed.
• As a result; multiple copies of transgenes are integrated
at random locations in the genome of the transgenic
individuals.
• The transgenes in many transgenic individuals are also
transmitted through the germline to subsequent
generations.
7
Note; If the transgenes are linked with
functional promoters, expression of
transgenes as well as display of change in
phenotype is expected in some of the
transgenic individuals
• Questions to be asked about any
transgenic animal are;
• how many copies it has of the foreign DNA
(varies 1-50)
8
• where these copies are located [usually multiple copies
are integrated into a tandem array (arranged adjacent
to each other) into a single chromosomal site]
• whether they are present in the germ line and inherited
in Mendelian manner.
• can the gene be expressed independently? i.e does
the regulatory elements function independently
• are transfected genes expressed with the proper
developmental specificity?
• A good result if we obtain 15% of the animals to be
transgenic.
• In the progeny of the infected animal, the expression of
the donor gene is extremely variable and that could be
dependent on the place of integration of the new DNA.
9
Transgenesis; Methodology
• Transgenic technology has been developed and
perfected in the laboratory mouse. Since the
early1980’s hundreds of different genes have been
introduced into various mouse strains. These studies
have contributed to;
• understanding of gene regulation
• tumor development, example introducing oncogenes
and observe the effect
10
• immunological specificity, example producing knockout
genes that are responsible for some immunological
aspects
• molecular genetics of development
• other biological interests such as examining the
possibility of using transgenic animals in the industrial
production of human therapeutic drugs.. etc.
11
Methods of gene transfer in animals
For transgenesis, DNA can be introduced into mice by
one of the following methods;
• Retroviral vectors that infects the cells of an early stage
embryo prior to implantation into a receptive female.
• Microinjection into the enlarged sperm nucleus (the
male pronucleus) of a fertilized egg
• Introduction of genetically engineered embryonic stem
cells into an early stage developing embryo prior to
implantation into a receptive female.
• Transfer of diploid somatic nuclei into an enucleated
oocyte.
12
Retrovirus-Mediated Gene Transfer
• The most useful vectors for the purpose of gene
isolation are those that lend themselves to the
production of libraries consisting of overlapping
fragments of genomic DNA, ideally encompassing
the entire genome several times.
• Exmaple; bacteriophage λ genomic library of 106
viruses each containing on average 20 Kb of
DNA, represents 6-7 copies of the entire mouse
genome and the probability that each gene is
represented is very high.
• Retroviruses can be used for the transfer of
foreign genes into animal genomes.
•
13
• This can best be done at 4-16 cell stage embryos.
However, it can be done up to midgestation, but with
incomplete infections i.e low infectivity rate.
• Immediately following infection, the retrovirus produces
a DNA copy of its RNA genome using its reverse
transcriptase.
• Completion of this process requires that the host cell
undergoes the S phase of the cell cycle. Therefore,
retroviruses effectively transduce only mitotically active
cells.
• Modifications to the retrovirus frequently consist of
removal of structural genes, such as gag, pol, and env,
which support viral particle formation.
14
• Additionally, most retroviruses and complementary lines
are ecotropic in that they infect only rodents, such as
rats and mice, and rodent cell lines rather than humans.
• The DNA copy of the viral genome, or provirus,
integrates randomly into the host cell genome, usually
without deletions or rearrangements.
• Because integration is not by way of homologous
recombination, this method is not used effectively for
site-directed mutagenesis.
• Very high rates of gene transfer are achieved with the
use of retroviruses.
15
Table of common vectors used for such purpose
•
•
•
•
•
Vector
Origin
Insert size range
Multicopy plasmids
multicopy plasmids up to 20 kb
Lambda vectors
Bacteriophage λ
up to 30 kb
Cosmid
Bacteriophage λ
up to 40 kb
P1 artificial chrom
Bacteriophage P1
80-90 kb
Bacterial artificial chrom. Large Bacteria plasmid 100300 kb (F factor)
Yeast chrom. (YAC)
Yeast chromosome 100-1000 +
kb
+ means indefinite.
16
Disadvantages of this method include:
• Low copy number integration.
• Additional steps required to produce
retroviruses.
• Limitations on the size of the foreign DNA insert
(usually 9 to 15 kb) transferred.
• Potential for undesired genetic recombination
that may alter the retrovirus.
• High frequency of mosaicism.
• Possible interference by integrated retroviral
sequences in transgene expression.
17
• The genome of the retroviral strain can be integrated
into the same nucleus as the transgene. This means
that the virus itself could be produced by the
transgenic organism and create a problem especially
if the animal will be used for production of food.
• Also the provirus attracts methylation which possibly
in conjugation with other mechanisms disables its
expression when it passes through the germ line.
• Due to this, and to the availability of other alternative
methods, the retroviral vector method is rarely used
for producing transgenic animals that have a
commercial potential.
18
DNA Microinjection Method
Because of the disadvantages of the retroviral vectors, microinjection
of DNA is currently the preferred method for producing transgenic
mice.
• First - you need the gene of interest in the proper form. A linear
transgene construct is made, which contains:
– the structural gene of interest, with introns
– a strong mouse gene promoter and enhancer to allow the gene to be
expressed
– vector DNA to enable the transgene to be inserted into host DNA
• The immature female mice will be induced to superovulate by
sequential administration of FSH/LH and HCG and mated to fertile
males. One-celled embryos are flushed from the oviducts and
placed in a drop of medium and viewed by phase-contrast or
interference microscopy.
19
This procedure has the following steps;
• The number of available fertilized eggs that
are to be inoculated are increased by
stimulating donor females to superovulate.
• This can be done by
– Giving the mice an initial injection of pregnant
mare’s ( an adult female of horse or related
mammal) serum
– Another injection about 48 hours later of human
chorionic gonadotropin (hCG). By this protocol the
female produces about 35 eggs instead of the
normal number of 5-10.
20
• These females are mated, then sacrificed and the fertilized eggs
(oocytes) are flushed from their oviducts and recovered.
• Eggs are treated with hyaluronidase to remove adherent follicle
cells.
• Unfertilized eggs are discarded
• The eggs are inoculated immediately with the transgene, briefly;
– embryo at the pronuclear stage is held in place by suction.
– a micro needle loaded with a suspension of plasmid DNA will be
prepared.
– It is introduced through the zona pellucida and plasma membrane into
the most accessible pronucleus (usually the male) and
– several hundred molecules of the recombinant DNA are injected in a
volume of approximately 1 picoliter (p1).
– on a good day several hundred eggs can be injected.
• The male pronucleus can be located by using dissecting
microscope and the eggs then can be maneuvered, oriented and
held in place while the DNA is microinjected.
21
oocyte
Pippet
Micro needle
22
23
• After inoculation, 25-40 eggs are implanted
microscopically into a foster mother who has
been made pseudo-pregnant by being mated to
a vasectomized male so that none of the eggs
of the foster mother will be fertile therefore, the
foster mother will deliver pups from the
implanted fertile eggs three weeks after the
inoculation.
• After birth, the presence of foreign material is
studied by DNA hybridization with appropriate
probes or PCR.
• A transgenic mouse can be mated to another to
produce transgenic homozygous transgenic
animal.
24
More details on gene transfer.
25
Gene Transfer to Animal cells
Dr. Ziad Jaradat
• There are four major strategies for gene
transfer to animal cells, two of which are
considered biological mechanisms using virus
(transduction) or bacterial that invade animal
cells (bactofection), those methods involve
infection while the other two are chemical and
physical methods which do not involve infection
thus termed transfection.
• Virus (transduction); the transferred gene
represents part of the viral genome
• Bacteria (bactofection); the gene will be
transferred as a plasmid
•
Chemical transfection
; DNA will be taken from the surroundings
when the DNA is presented as a synthetic
complex either as;
–
–
a complex with overall positive charge, allowing
it to interact with negatively charge cell
membrane and promote uptake by endocytosis
as lipophilic complex that fuses with the cell
membrane and deposits the transgene directly
into the cytoplasm.
Physical transfection;
•
•
in this method, naked DNA is deposited
directly into the cell by exploiting a physical
force. This includes;
•
•
•
•
microinjection
particle bombardment
ultrasound
electroporation
Which ever method used, the result is called
transformation which is a change in the
recipient cell’s genome caused by the acquired
transgene.
Chemical Transfection Techniques
• Calcium phosphate method; involves the
formation of a fine DNA/calcium phosphate
co-precipitate which first settles on the cells
and then internalized by endocytosis.
• The precipitate must be formed freshly at
the time of transfection. The DNA escapes
and reaches the nucleus and can be then
expressed. Since the cells must be coated by
the calcium complex, monolayers of cells must
be used for maximum efficiency. However,
this method gives only 1-2% transfection
efficiency.
•
Transfection with polyplexes; this is more efficient
than that with calcium as it gives more uniform
particle size. Polyplexes are a polycationic
compounds that form soluble complexes (polyplexes)
through spontaneous electrostatic interaction with
DNA.
•
This method is adapted as a plasmid DNA transfer.
The efficiency of the method can be increased by
exposing cells to osmotic shock or treatment of cells
with chloroquine.
•
Anew generation of the polycationic compounds has
been developed such as poly-L-lysine and synthetic
poly amines, polyethelylenimines and dendrimers
(highly complex molecules built in layers from a
central initiator such as ammonia or ethylenediamine).
Transfection with liposomes and lipoplexes
•
•
This can be done by packaging the DNA inside
a fusogenic phospholipid vesicle which
interacts with the target cell membrane and
facilitate DNA uptake.
Briefly, bacterial cells will be transformed
with suitable plasmid vector and treated with
chloramphenicol to amplify plasmid copy
number.
• The cells will be treated with lysozyme to
remove cell wall, resulting in protoplasts that
will be centrifuged gently onto a monolayer of
mammalian cells to promote fusion among them
using polyethylene glycol.
• Using lyposomes is more commonly used for
this type of transfection. This method is
commonly known as lipofection. This method is
far more efficient than chemical transfection
method. With this method up to 90% of cells in
culture dish can be transected.
Physical transfection methods
• Electroporation
• Ultrasound
both methods create transient pores in the cell
Electroporation
• is a physical transfection technique involves
creating transient nano-meter size pores in
the cell membrane by exposing cells to a brief
pulse of electricity. The most critical
parameter is the intensity and duration of
electrical pulse.
• Electroporation can be used for in vivo gene
transfer particularly for surface or near
surface tissue such as skin, muscle and
certain tumors or even internal tissues such
as liver. This can be achieved by direct
application of electrodes to the skin following
shaving and mild abrasion with the DNA being
injected into the skin before electroporation.
Ultrasound transfection
• Involves the exposure of cells to a rapidly oscillating
probe such as the tip of sonicator. In this method the
application of ultrasound waves to a dish or cells or a
particular tissue results in the formation and collapse
of bubbles in the liquid, including the cell membrane, a
process known as cavitation.
• The transient appearance of such cavities allows the
DNA to cross the membrane into the cytoplasm. This
method can be used both for in vivo or in vitro as the
plasmid DNA is left structurally intact. As for
electroporation, the DNA will be injected and then the
ultrasound will be applied.
DNA microinjection
Direct transfer of DNA into the cell without
a carrier is called DNA microinjectin. This
can only be done for only few cells at a
time. This technique is used mainly for
large cells such as oocytes, eggs and the
cells of early embryos. The DNA can be
directly injected into tissues, such as skin,
muscle or internal organs or it can be
injected into the blood.
• The process is remarkably efficient. Up to 6066%% of the embryos survive injection and up to
25-30% of the embryos transferred to the oviduct
survive to birth and about 25% of pups are
transgenic (transgenic founders). Thus, from 1000
inoculated fertile eggs, 30-50 (3-5%) transgenic
pups are produced.
• The injected DNA gets incorporated at random
sites within the genome and often multiple copies
are incorporated at one site, therefore, not all the
transgenic animals will have the desired traits.
38
Particle bombardment
• is another direct delivery method initially
developed for the transformation of
plants. This method involves coating
small metal particles with DNA and
accelerating them into target tissues
using a powerful force such as the blast
of high pressure gas or an electric
discharge through a water droplet. In
animals, this method is used for tissues
such as skin cells in vivo rather than
cultured cells.
Bacterial vectors for gene transfer
The exploitation of living bacteria for gene
transfer is central to the genetic manipulation
of plants. Agrobacterium tumefaciens and its
close relatives have been used for 20 years to
generate transgenic plants.
• Recently, A. tumefaciens has been used to
transfer DNA to human cells. The protoplast
fusion technique can be considered as highly
efficient form of bactofection. However, this
type needs human intervention, while the A.
tumefaciens does not require human
intervention.
How does the bacterial transfer of DNA happen?
• The bacteria invades the host animal cells and
undergo lysis within them releasing their plasmid
DNA. Example of these bacteria including
Salmonella species (lysis occurs in the
phagocytic vesicle), Listeria monocytogenes and
Shigella flexneri (lysis occurs for these two
species after they escape from the vesicle).
•
The plasmid DNA then finds its way to the
nucleus where it gets incorporated with the
cell’s genome and gets expressed.
•
Contrary to the above mentioned bacteria, A.
tumefaciens does not invade the cell, instead,
it attaches itself to the outside surface
followed by conjugation.
Viruses That are used as gene transfer
vectors
• Virus particles have a natural ability to adsorb
to the surface of the cells and gain entry. This
can be exploited to deliver recombinant DNA
into animal cells.
• Several classes of viruses has been used for
gene therapy and at least 8 has been used in
clinical trials. Transgenes may be incorporated
into viral vectors either by addition to the
whole genome or by replacing one or more viral
genes. This can be done by ligation or by
homologous recombination.
• If the transgene replaces a none essential gene
the vector is described as helper-independent
• If it replaces an indispensable gene, then this
vector will be helper dependent.
• It is generally recommended to use vectors from
which all viral coding sequences has been deleted
such vectors are described as fully deleted or
gutted or gutless vectors. These vectors contain
just the cis-acting elements required for
packaging and for genome replication.
Advantages of such vectors
• high capacity for foreign DNA
• because no viral gene products are made, the vector
has no intrinsic cytotoxic effects.
Adeno virus
• These are viruses with a linear, double stranded
genome, of approximately 36 kb. These are used
frequently due to certain advantages;
–
–
–
–
stability
high capacity for foreign DNA
wide host range including none-dividing cells
Ability to produce high titer stocks up to 1011 pfu/ml.
Adeno-associate virus (AAV)
• These viruses are not genetically related to
adenovirus but is so-called because it was first
discovered as a contaminant in an adenovirus
isolate.
• The AAV is a single stranded DNA and is a
member of the parvovirus family.
• It is naturally replicating deficient, thus it
requires the presence of another virus such as
adenovirus to complete its infection cycle. AAV
replicates lytically and produces thousands of
progeny virions.
• The dependence of AAV on a heterologous
helper virus such as adeno virus provides an
unusual degree of control over vector
replication making AAV one of the safest
vectors to use for gene therapy.
• Other advantages of this viral vector is the
wide host range that it exhibits including none
dividing cells.
• The AAV genome is small (5 kb) and
comprises a central region containing rep
(replicase) and cap (capsid) genes flanked by
145 kb inverted terminal repeats.
• Foreign DNA replaces the cap region and gets
expressed by indigenous promoter. However rep
proteins might interfere with the expression
process thus responsible for some of the
cytotoxic effects of the virus.
• New vectors are designed with the deletion of
the rep and cap genes and only utilizing the
repetitive sequence which are the only
elements required for replication, transcription
and proviral integration.
• AAV vectors have been used to introduce genes
efficiently into many cells including liver,
muscle, and neurons.
Baculovirus vectors (BV)
• Baculovirus promote high level of transgene
expression in insect cells but can also infect
mammalian cells.
• Have rode shape capsid and large doublestranded DNA genomes. They productively infect
arthropods particularly insects.
• BV vectors are used mainly for high–level
transient protein expression in insects and insect
cells.
• These viruses produce a protein called
polyhedron at a very high expression level yet
this protein is not necessary for the infection
process therefore it can be replaced with
foreign DNA that can be expressed at high
levels (up to 1 mg / 106 cells) under the control
of the endogenous polyhedron promoter.
• The highest amount of expressed proteins was
initially achieved when the transgene was
expressed as a fusion protein by incorporating it
with the first 30 amino acids of the polyhedron
protein.
There are two types of BV that are
used as vectors;
– Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV); infects
mainly insects
– Bombyx mori nuclear polyhedrosis virus
(BmNPV); infects insects silkworms and
is used for production of recombinant
proteins in silk worm larvae.
• One problem of expression of mammalian
proteins in insects is the different types of
glycosylation. Thus as a solution, is to exploit the
indefinte capacity of BV vectors to co-express
multiple transgenes and thus modify the
glycosylation process in the host by expressing
appropriate glycosylation enzymes along with the
transgene of interest.
• Example this strategy was used to co-express
fowl plague hemagglutinin and beta-1,2-Nacetylglucosaminyl-transferase resulting in the
synthesis of large amounts of hemagglutinin
correctly modified with N-acetylglucosamine
residues.
• The construction of baculovirus expression vectors
involves inserting the transgene downstream of the
polyhedron promoter. This is normally achieved by
homologous recombination using plasmid vector carrying a
baculovirus homology region.
• Baculoviruses can also be used for the delivery of foreign
genes to viruses to produce a cell model or animal models
for certain diseases. Example the hepatitis C virus does
not infect cultured cells, but a hybrid baculovirus
containing the entire HCV genome can initiate an HCV
infection.
Genotyping Transgenic Mice by PCR to Screen for
Potential Founders
• This is the test method of mice for the presence of the
transgene by PCR.
• Since we know the sequence of the gene that was
inserted into the male pronucleus, we could determine
if the mouse contains the transgene of interest, by
performing PCR.
• Tail biopsies from potentially transgenic mice will be
obtained 5 weeks after injecting eggs (3 weeks
gestation time and 2 weeks of post-natal growth). The
investigator then extracts DNA from the tail tips and
tests for the transgenic by PCR. How?
55
• By designing a set of primers that are taken from the
transgene sequence and using them in a regular
PCR to amplify the gene of interest if found.
• Now if the mouse is a transgenic mouse, then a
PCR product corresponding to a known size will
appear in the gel. But if the mouse is NOT a
transgenic one, there should be NO PCR product
corresponding to that size.
• In addition, to evaluate the stability of the insert,
some markers has to be checked and the best ones
are the ones that can be assayed readily such as
observing the new phenotype of the progeny.
56
To determine the number of copies and places,
Southern Blotting Analysis will be done;
• When pups are 6 weeks old, Southern blot
analysis should be done to determine how many
copies of the transgene were integrated, how
many chromosomal sites the transgene inserted
into, to verify transgenic status and to determine
if the transgene is intact.
• With this information, transgenic founders with a
good chance of transmission (at least 5-10
copies) of an intact transgene in a single insertion
site can be selected for intensive breeding.
(Figure 1 ).
57
58
• One of the problems is that when DNA
is micro-inserted, randomly some parts
of it will replace some genes in the
mouse, and thus might inactivate them.
• Depends on which gene is inactivated,
a damage to the progeny might occur.
59
Engineered Embryonic Stem Cell Method
• In this method, cells from the Inner Cell Mass
(ICM) of early embryos blastocysts (a stage of a
developing mouse embryo) will be used.
• These cells can be grown in cell culture and still
retain the capability of differentiating into other
cell types including germ line cells after they are
introduced into another blastocyst embryo.
• Such cells are called pluripotent (multi)
embryonic stem (ES) cells. These cells can be
easily manipulated by genetic engineering
without changing their pluripotency.
60
Steps of the procedure;
1. Obtain fertilized eggs (pre-implantation zygotes) from a
pregnant mother mouse as described above.
2. Grow zygotes in culture until day 3.
3. Harvest the Inner Cell Mass (ICM) from 3 day old
blastocysts.
4. Culture the Inner Cell Mass (ICM) on feeder cells to
develop Embryonic Stem (ES) Cell lines.
61
5. Create transgenic ES cells by microinjection or by
introducing cells briefly to an electrical potential that
disrupts cell membrane thus allows the entrance of
DNA containing the transgene that was constructed
with the genes of interest.
• In this method a functional transgene can be integrated
in the place of a dispensable gene in the genome of the
ES cell.
62
6. Inject the transgenic ES cells into the blastocoele (fluid
filled cavity of the mass of cells) of a new 3-day old host
blastocyst.
• The injected ES cells combine with the host ICM and
contribute to the developing embryo.
• The first generation offspring are chimaeras - they have
somatic cells composed of both transgenic ES cells and
host cells
• And also have germ cells composed of both transgenic
ES cells and host cells
• Usually a coat color gene is used in the transgene
construct as a visual marker to facilitate the quick
detection of the transgenic (chimaeric) pups.
63
7. The transgene, if in the germ cell lineage, can be
transmitted to offspring and homozygous transgenic
lines can be constructed.
• After transfection of ES cells in culture with the DNA
vector;
– Some cells will have DNA integrated at none-target
(spurious) sites
– Some cells will have DNA integrated at target (correct) sites
– Some cells will not have any DNA integration
64
How to enrich DNA integration at the specific sites?
A procedure called positive/negative selection is
implemented.
•
This procedure used positive selection for cells did
not accept the DNA inserts and negative selection
for cells who have DNA integrated any where in
their genome.
•
–
In this procedure, a construct will be prepared and
should contain the following;
Two blocks of DNA sequences (HB1 and HB2) that are
homologous to separate regions of the target site.
65
• The trans gene, TG that will confer a new
function on the recipient
• Neor, a DNA sequence that codes for an
enzyme that inactivates neomycine and its
relatives such the drug G418 which is
lethal to mammalian cells
66
•
Two different genes for the thymidine kinase
(tk1 and tk2). These enzymes phosphorylates the
nucleoside analogue called gancyclovir.
DNA polymerase fails to discriminate against the
resulting nucleotide and inserts this nonfunctional
nucleotide into freshly-replicating DNA. So
gancyclovir kills cells that contain the tk gene.
•
Now the arrangement of these sequences is key to
the positive and negative selection procedure.
67
Possible results;
•
Most cells fail to take up the vector; these cells will be killed if
exposed to G418 as the neo gene will not be incorporated.
(positive selection).
•
In a few cells: the vector is inserted randomly in the genome.
In random insertion, the entire vector, including the tk genes,
is inserted into host DNA. These cells are resistant to G418
but killed by gancyclovir. (Negative selection).
•
In still fewer cells: homologous recombination by double
crossover at target sites occurs. i.e Stretches of DNA
sequence in the vector find the homologous sequences in the
host genome and the region between these homologous
sequences replaces the equivalent region in the host DNA.
•
Therefore, tk genes will be excluded and cells survive both
G418 and gancylovir as only the neo and the trans genes are
included.
68
69
• Now by this method ES cells that carry the target site
will be enriched several thousand fold, thus better
chances of producing a transgenic animal with the
desirable characters.
• By this method, ES cells that contain the target, are
identified and cultured for propagation.
• Embryonic stem cells carrying an integrated transgene
can be cultured and inserted into blastocyst stage embryo
and these embryos can then be implanted in
pseudopregnant foster mother.
70
• Transgenic lines can then be established by first
mating founder transgenic mouse to animals from
the same strain and then crossing transgenic litter
mates to create a homozygous transgenic animal.
• Unfortunately pluripotent ES cells comparable to
those of mouse were not found in cattle, sheep,
pigs or chickens.
71
Scientific and medical applications of the ES cells
method of transfection
• This route has been usually employed to; inactivate a
gene, alter it, or replace its protein coding region
with a reporter (a coding unit whose product is
easily assayed. It may be connected to any promoter
of interest so that expression of the gene can be
used to assay promoter function).
• Main application of ES cell transgenic mice are to
medicine and pure science including;
– Improve understanding of all aspects of healthy animal
– Understanding therapeutic approaches
– Understanding biochemistry and physiology particularly;
mammalian development, neurobiology, learning and memory.
72
Nuclear Transfer Method (non-transgenic method)
• In this process the sheep Dolly was generated from an
enucleated (nucleus was removed) egg into which the
nucleus from a cultured somatic cell of a mature
sheep has been introduced.
Method
• Oocytes are recovered from animals between 28-33
hours after injection of gonadotropin releasing hormone,
• Oocytes are recovered in PBS containing 1% FCS and
transferred to a new media containing 10% FCS and
incubated at 37° C.
• Nucleus is removed manually from an unfertilized
oocyte as soon as possible
73
• The somatic cell has to be in a non-dividing stage
(G0) why ? ; this can be done in culture by depriving
it of external stimuli that provokes growth. How?.
Read the provided article
• A non-dividing somatic cell is placed in contact with
the oocyte and the two are fused together by
applying an electrical potential which also activates
the egg thus, mimicking the process of natural
fertilization.
• The result of this hybridization is an activated oocyte
with two chromosome sets (from the diploid somatic
cell).
74
• Usually, the cytoplasm of normal oocyte contains proteins
and RNA molecules that are required for the early stages
of development but in this case, the cytoplasm of the
somatic cell contains a whole set of genes that are
reprogrammed to take control over the developmental
program in the same way as the genes of the normal
embryo.
• Effect of age; it was found that cells obtained from
fetuses and new borne donors are more efficient in
nuclear transfer while clones derived from adult cells
show more abnormalities.
• Why? It could be due to the fact that somatic cells of
adult animals have accumulated more mutations or they
are more differentiated than fetal cells thus are more likely
to fail the full term development.
75
Applications of Nuclear Transfer;
• Nuclear transfer has applications outside the
field of transgenesis such as propagation of an
animal with a particularly desirable set of genes.
• Since propagating the transgenic animals are not
easy and normally goes with risks, the nuclear
transfer can therefore, be used for propagating a
successful transgenic animal making a whole herd
of that animal !!! ….. Prohibitively expensive.
76
Applications of transgenic animals
Transgenic mice
Transgenic mice can be used for;
– As test subjects to determine the effectiveness
of potential therapeutic agents
– Although mice are far from humans, some times
they can serve as models for human diseases.
77
Specific Applications of Transgenic Mice
Transgenic Mice in Oncology
• The study of transferred oncogenes has always been
hampered by the fact that cell lines in culture have
already been transformed to an abnormal phenotype.
• The ability to insert oncogenes or proto-oncogenes
into embryos and to study their effects in normally
differentiating cells of an intact organism has
circumvented this problem. Results of such studies
have made an enormous contribution to our
understanding of neoplastic diseases and its
relationship to aberrant gene expression.
78
Transgenic Mice as Animal Models of Human Diseases
Animal models for human illnesses are useful for studying
the pathogenesis of diseases as well as for developing
and testing new therapies. Human diseases can be
induced in transgenic mice by expression of
transferred genes, or by insertional disruption of
endogenous sequences.
Some examples of models created by transgene
expression are listed below.
• Hepatitis B is a human disease that lacks a readily
workable animal model. Introduction of the HBSAg
gene into mice results in transgenic mice that mimic the
carrier state with production of HBsAg in the liver but
with an absence of disease
79
Transgenic Mice as Models for Gene Therapy
• Genes can be inserted into transgenic animals and
function to alleviate disease states, such model systems
can be of great importance in improving our
understanding of the potential for gene transfer as an
approach to treatment of diseases.
• Mice with growth hormone deficiency are markedly
reduced in size and males suffer from infertility.
Introduction of the growth-hormone gene into these
animals leads to growth which exceeds that of normal
animals and restores male fertility.
• However, the pattern of release of growth hormone that
results from transgene function is apparently
inconsistent with female fertility. i.e does not restore it.
80
81
Another Example
• Insertion of either the mouse or human β-globin gene
can reduce the severity of β-thalassemia in mice. In
these experiments, the product of the human globin
gene was able to associate effectively with the mouse
A chains, and it actually functioned better than the
transferred mouse gene β-globin in reducing severity
the thalassemic state.
• Mice with a deficiency in gonadotropin-releasing
hormone (GnRH) are infertile and exhibit profound
perturbations of their reproductive endocrine
functions. Cloning of the GnRH gene and its transfer
into mice has resulted in restoration of normal
endocrine function and in fertility.
82
Alzheimer’s disease model
• Alzheimer’s disease is a degenerative brain disorder that is
characterized by the progressive loss of both abstract thinking
and is accompanied by personality change, language disturbance
and a slowing of physical capabilities.
• The brain of those patients accumulate within the body of the
neurons a dense material called senile plaques.
• The principal component of senile plaques and amyloid bodies is
a 4-kDa protein called βA4 (or β protein). This protein is the
product of an internal proteolytic cleavage of the β- amy1oid
precursor (APP).
• Researchers have found that some strains of mice produce
senile plaques during their life span, whereas others do not.
Thus, the later (none producers) strains are important for
forming transgenic mice that carry and express a transgene
encoding the βA4 portion of APP which might provide a model
for studying the molecular basis of Alzheimer’s disease.
83
Importance of such experiment: this type of mice
can be used for a precise determination of the
mechanism of Alzheimer’s disease and probably for a
treatment scheme.
• One of the vectors that has been constructed for
modeling Alzheimer’s disease in mice consists of ;
– A promoter region from brain specific virus ligated to a
portion of the human APP (β amyloid precursor protein) gene
that encodes the last 100 amino acids at the C terminus of
APP, which includes the βA4 amino acid sequence.
– Transgenic mice were established with this construct, and
expression of the transgene was confined to neurons of the
brain.
– Immunocytochemical studies showed that the brain of
transgenic mice accumulated βA4 protein that was derived
from the transgene. How?
84
• This screening can be done using anti- βA4
antibody that is conjugated to a dye which makes
it visible either to the naked eye or under special
microscope such as fluorescence.
*******
• Alternatively, ES cells that have a site-directed
mutated APP gene could be used to establish a
transgenic line that might mimic Alzheimer’s
disease more precisely.
• Transgenic mice have also been used as models
for expression systems that are designed for
secretion of the product of a transgene into milk.
85
Another example (CF)
• Another example of the usefulness of the transgenic
mice is the production of large quantities of
authentic cystic fibrosis trans-membrane regulator
(CFTR) that are needed to study its function and
possibly formulate potential therapies for treating
cystic fibrosis.
• CFTR normally acts as a chloride channel but when its
function gets altered, cystic fibrosis occurs and it
will be characterized by the accumulation of mucus
into the lungs and pancreas.
86
What is Cystic fibrosis?
• It is the most common lethal human hereditary disorder,
occurring once in every 3,000 births.
• It affects the lung, intestinal tract and liver, with thick
mucus, chronic airway infections and inflammation beginning
in early childhood and leading to progressive loss of lung
function.
While the life expectancy of these children is double what it
was, they are still only expected to live to 40. The
underlying defect is in a gene that codes for a substance
that regulates protein secretion across a cell membrane,
but infection also plays a major role.
All existing therapies only alleviate the symptoms by reducing
infection and mucus.
87
To get large amounts of CFTR;
• A full length CFTR cDNA sequence was cloned into
the middle of a defective goat β-casein gene
• The construct retained the promoter and the
termination sequences of the goat β-casein gene.
• The β-casein gene is then actively expressed in
mammary glands during lactation producing the βcasein which is the most abundant protein in the
milk.
88
• Now, transgenic mouse lines carrying the CFTR sequence
under the control of the β-casein gene regulatory
sequences were established.
• The product is milk from transgenic females contained the
CFTR protein bound to the membrane of fat globules.
• This is a model, however, to obtain mega quantities of this
protein, a construct has to be introduced into a larger
animal such as sheep, cows or goat.
89
Antisense Genes in Transgenic Mice
• Another method for negating gene function involves the use
of antisense transcripts.
• When genes are cloned in reverse orientation with respect to
the promoter, RNA may be produced from the non-coding
strand.
• This RNA, presumably by forming a heteroduplex with the
sense RNA, can block translation of cytoplasmic mRNA.
• Thus, antisense genes can be used to obliterate (wipe out)
production of proteins from specific genes in transgenic
animals.
• The feasibility of this approach has recently been
demonstrated by the transfer of an antisense construct of
the gene for myelin basic protein (MBP) into mice.
Interference with the production of MBP resulted in
dysmyelination. Although this research is still its infancy, it
has great potential for future experiments.
90
Transgenic cattle
• If the mammary gland is to be used as
a bioreactor, then dairy cattle are the
likely candidates for transgenesis as
they produce about 10,000 liters of
milk/year with 35 gm protein/liter.
91
Protocol to produce transgenic cattle (Figure 15-9)
•
•
•
•
•
•
•
•
•
collecting oocytes from slaughterhouse –killed cows
in vitro maturation of these oocytes
in vitro fertilization with bull semen
centrifugation of fertilized eggs to concentrate the yolk so
that male pronuclei will be seen under the dissecting
microscope.
microinjection of input DNA into male pronuclei
in vitro development of embryos
embryo implantation into a recipient foster mother
DNA screening of the offspring for the presence of the
transgene.
When this procedure was done only two transgenic calves were
produced from a starting pool of 2470 oocytes which means
that the procedure is feasible but in efficient in this format.
92
Goals of producing transgenic cattle
•
To change the constituents of milk. For example the amount of
cheese produced from milk is directly proportional to the amount of
k-casein content of the milk so if a transgene is constructed to
produce milk with higher amounts of k-casein, then the production of
cheese will increase proportionally.
•
Production of transgenic cows with modified genes to produce lactose
free milk could solve the problem of those who have lactose
intolerance.
•
For livestock in general, attempts to produce animals with inherited
resistance to bacterial, viral, and parasitic disease is a goal. Example
of major diseases that affect the livestock are mastitis in cows,
neonatal dysentery in swine, fowl cholera.
•
If the basis of each of these is a single gene that will be responsible
for the resistance, then it might be possible to produce transgenic
animals that carry this gene.
93
Other alternative, is the production a transgenic
animal with inherited immunological protection.
A number of candidate genes that contribute to the
immune system such as Major histocompatibility
genes, T-cell receptor genes, lymphokine genes are
under study to evaluate this potential.
But the most favorable preliminary results to date
comes from research in which the genes encoding
the heavy and light chains of a monoclonal antibody
(MAb) have been transferred to mice, rabbits and
pigs.
94
• By this introduction of MAb, these
animals will have an endogenous source of
MAbs with predefined specificity toward
certain pathogen, thus eliminates the need
for immunization. This concept is called In
vivo immunization.
95
• Example; the genes for the immunoglobulin chains
of a mouse MAb that are specific to “ 4-hydroxy3-nitrophenylacetate were cloned in a tandem and
microinjected into fertilized egg of mice, rabbits
and pigs.
• In each case MAb activity was found in the serum
but the concentrations of the antibodies were low
which could be due inheritable problems of the
construct, thus a new construct should be tested.
96
Transgenic Sheep
• Transgenesis research with sheep, goat or pigs has
concentrated in the most part on utilizing their
mammary glands as bioreactors for production of
pharmaceutical proteins.
Example; Production of transgenic sheep that
produces anti-trypsin in their milk; This protein is
a potential treatment for cystic fibrosis.
The Technology
• PPL Therapeutics transfers genetic material from
one organism to another using the same technology
it used to produce "Dolly the Sheep", a process
called somatic cell nuclear transfer.
97
Steps of the procedure
Genes are Modified: A single cell from a sheep is
modified to include the human gene for the protein
alpha-1 antitrypsin. However, the gene must only
turn on in the mammary glands so that the protein
only appears in the sheep's milk.
• Before the sheep DNA is modified, the human gene
is fused to the promoter gene for beta-lactoglobulin.
The human gene will only be expressed when the
beta-lactoglobulin is turned on, and this only happens
in the milk-producing mammary glands.
Injection : The nucleus, containing the modified DNA,
is removed from this cell and injected into the
enucleated fertilized sheep oocyte. Or the modified
somatic cell could be fused to the enucleated
oocyte.
98
Implantation of embryos: The fertilized sheep embryo is
implanted into a surrogate mother for the rest of its
pregnancy.
Lactation: Upon giving birth to a lamb the mothers (ewes)
produce milk (lactated). Beta lactoglobulin production
started during lactation, so did production of human alpha-1
antitrypsin. The rams also contain the required gene but it is
not active, although it can be passed to their offspring. The
alpha-1 antitrypsin protein that is expressed in the milk can
be extracted and purified.
Next Generation: The newborn lambs were screened for
presence of the gene (by DNA analysis of tail tissue or blood
from the jugular) and mated when mature.
The production flock was started from semen from two
transgenic rams brought to New Zealand in 1996.
Conventional New Zealand ewes were inseminated and some
of the resulting lambs were transgenic. Embryos from
transgenic animals were transferred to surrogate mothers.
99
The modified gene is shown to be stable (i.e has
been transmitted faithfully from parent to
offspring). How can we judge that?
• Homozygotes are as healthy as heterozygotes;
this shows that the gene has not inserted into an
essential part of their genetic material - insertion
into other parts of the DNA would lead to death
of the offspring
• The human protein secreted in the milk has been
consistent in quantity and quality
100
The Benefits
• The most obvious benefit from this
research is:
• Production of a treatment for cystic
fibrosis. How? See next slide
101
Human alpha-1 antitrypsin is currently derived from blood plasma and
administered intravenously at 60mg/kg once a week. The difficulties
with this treatment are:
1. Cost - treatment for an individual costs $40,000 per year
2. Availability - the protein is produced in plasma at a concentration of
about 1.5 g/L and obtained from healthy donors.
3. Contamination - any extraction of material from blood carries risk of
contamination from other diseases such as HIV, new variant
Creuzfeldt-Jakob Disease (BSE) and Hepatitis B.
4. Efficiency - using transgenic animals produces far greater quantities of
the protein at lower cost in the long term, this research will also
provide further benefits:
• Provide techniques for producing other disease-fighting drugs
• Provide techniques for incorporating medicines in foods
• Help scientists to understand how milk protein is produced and modified
Transgenic-derived proteins were glycosylated and had biological activities
comparable to those extracted from human sources.
102
Goats and Pigs
Generally the production of transgenic goats and pigs is
similar to that for sheep however, there are some
differences in that the;
– expression of transgenes in the mammary glands of sheep or
goats had no ill effects on either lactating female or nursing
progeny.
– While the transgene for bovine growth hormone-under the
control of the metallothionine promoter- when introduced
into pigs, several adverse results were observed;
•
•
•
•
•
•
Gastric ulceration
Kidney dysfunction
Lameness
Inflammation of the lining of the heart
Swelling of the joints
Susceptibility to pneumonia
103
Transgenic Birds
• Avian ova are normally fertilized approximately 30
minutes after ovulation. Cell division occurs in the
oviduct for approximately 20 hours before ovi
position. At this time, the embryo is comprised of
approximately 60,000 pluripotent cells, which are
collectively called the blastoderm.
• The presence of a large yolk and multiple pronuclei
makes direct microinjection of DNA impractical.
• Therefore, DNA microinjection into fertilized bird
eggs to produce transgenic strains is not possible;
104
• During fertilization in birds several sperms can penetrate
the ovum, instead of only one as in case of mammals.
• Therefore, it is not possible to identify the male pronucleus
that will fuse with the female pronucleus.
• Microinjection of DNA into cytoplasm is not enough for the
process to proceed as the DNA will not integrate into the
genome of the fertilized egg.
• The technique also would be difficult as the avian ovum
after fertilization become enveloped in tough membrane and
surrounded by large quantities of albumin and enclosed in
inner and out shell membranes.
105
• By the time the avian egg outer shell
membrane hardened, the developing
embryo (blastoderm stage) will be two
layers of 40,000 to 80,000 cells.
• At the moment no one has identified avian
specific embryonic stem cells so this
approach can not be used in birds. The
alternative is a procedure using engineered
cells from embryos. How??
106
Procedure
Plastoderm cells are removed from the donor chicken
These cells get transfected with cationic lipid
(liposome) transgene DNA complexes (lipofection).
The cells will be reintroduced into the subgerminal
space of embryos of freshly laid eggs. Figure 15-10
shows a schematic diagram of this procedure.
Some of the progeny will consist of a mixture of cells
with some cells from the donor but most from the
recipient, such mixture is called the chimera
* Lipofection: delivery into eukaryotic cells of DNA and RNA or other compounds
that have been encapsulated in an artificial phospholipid vesicle.
107
Now in some of these chimeras cells that were
descended from transfected cells may become part
of the germ line tissue and form germ cells.
Transgenic lines can then be established by rounds
of mating.
The proportion of chimeras can be increased to
enhance the probability of obtaining germ line
chimeras if the receiving embryos are irradiated
with a dose of 540-660 rads for 1 h prior to the
introduction of transfected cells.
Irradiation destroys some of the blasoderm cells
thus increasing the final ratio of the transfected
cells to the recipient cells.
108
What can we use transgenic chicken for?
To improve the genetic makeup of the existing strains
with respect to
resistance to avian viral and coccidial diseases,
better feed efficiency,
lower fat and cholesterol in eggs and
better meat quality.
The egg with its high protein content could be used as a
source of pharmaceutical proteins
109
Transgenic Fish
As natural fisheries become exhausted, production of this
source will depend more on the aquaculture. Production of
transgenic fish therefore become a primary objective.
To date, transgenes have been introduced by DNA
microinjection into the fertilized eggs of number of fish
species including;
Catfish , Crap, Trout , Salmon , Tilapia
In fish the pronuclei are not readily seen under the microscope
after fertilization, therefore, a linearized transgene DNA is
microinjected into the cytoplasm of either fertilized eggs or
embryos that have reached the 4 cell stage.
110
Now because fish eggs develop externally there will be no
need for implantation. Instead the development can be done
in the Temperature regulated tanks with a survival rate
from 35-80% and production of the transgenic fish ranges
from 10-70%. Same as in transgenic animals, the founder
fish can be mated and transgenic lines established.
In one study, a transgene consisting of the promoter region
of the antifreeze protein gene of the fish called ocean pout.
The growth hormone cDNA from salmon. And the
termination polyadenylation signals from the 3’ end of the
end of the antifreeze protein. This construct was injected
into eggs of Atlantic salmon.
111
Result; the transgenic salmon was larger and
grow faster than the none transgenic.
Eventually, genes for disease resistant,
tolerance to environmental stress, and other
biological features will be introduced into
fish in cold and warm waters.
112
Thank you…
Do you think you
have learned some
thing new?
113