Lec # 2. Animal Biotechnology

7/28/2019 Lec # 2. Animal Biotechnology

1/22

Animal Biotechnology


2/22

How are transgenic animals produced?

DNA microinjection

Introducing the transgene DNA directly into the zygote/Oocyte at an early stageof development.

No vector required.

Retrovirus-mediated gene transfer Infecting mouse embryo with a retrovirus which carry the new gene.

Using virus as a vector.

Embryonic stem cell-mediated gene transfer The blastocyst (inner layer of a fertilized egg) is harvested

Mixed with recombinant DNA.

Inserted back in the blastocyst.

Sperm-mediated transfer

Use ofLinkerprotein" to attach the desired genes for particular trait

The gene with linker proteins are the attach with the sperm which transfer thenew gene to oocyte during fertilization.


3/22

Refers to the process of using a glass micropipette to insert the desired

genetic materials into a single living cell.

It is a simple mechanical process in which a needle (0.5 to 5m in dia)

penetrates the cell membrane and/or the nuclear envelope.

The desired gene(s) are then injected into the nucleus and the needle isremoved.

Microinjection is normally performed under a specialized optical

microscope setup called a micromanipulator. The process is frequently

used as a vector in genetic engineering and transgenics to insert genetic

material into a single cell.

Microinjection can also be used in cloning of organisms & in the study of

cell biology and viruses.

DNA Microinjection


4/22

One of the first methods that proved to be effective in mammals(Gordon and Ruddle, 1981)

Direct microinjection of a chosen gene construct

A single gene or a combination of genes, from another memberof the same species or from a different species.

Into the pronucleus of a fertilized ovum.

The introduced DNA may lead to the over- or under-expressionof certain genes


5/22

The insertion of DNA is random process.

High probability that the introduced gene will not insert itself into aspecific site on the host DNA.

Manipulated fertilized ovum is transferred into the oviduct of arecipient female or foster mother.

Induced to act as a recipient by mating with a vasectomized male.

Applicable to a wide variety of species.


6/22

First Breeding Pair

Fertile male + superovulated female

Superovulated female: Immature female induced to

superovulate Pregnant mares serum on day 1

Human Chorionic Gonadotropin on day 3

Mated on day 3

Fertilized oocytes microinjected on day 4 with foreign DNAconstruct.

Microinjected oocytes are transferred to the oviducts ofsurrogate mothers at end of day 4.

Procedure for Producing Transgenic Mice


7/22

Second Breeding Pair:

Sterile male + surrogate mother

Sterile male produced through vasectomy (A surgical procedure formale sterilization and/orpermanent birth control. During the procedure the entry of sperms is blocked intothe seminal stream)

Surrogate mother must mate to become a suitable recipient ofinjected eggs

Mated on day 3

Microinjected oocytes from first breeding pairare transferred to

oviducts on day 4 Embryos implant in uterine wall of the surrogate mother and are

born 19 days later.



8/22

Third breeding pair:

Foster parents (Adoptive)

Fertile male + female mated to give birth on same day

surrogate mother. Serves as foster parent if caesarian section (Surgical delivery of an

infant through an incision in the mother's abdomen) is required for

surrogate mother.



9/22

Establishing transgenic mice

by DNA microinjection

Only 5% or less of the treated eggs

become transgenic progeny

Need to check mouse pups for DNA

(PCR or Southern), RNA (Northern or

RT-PCR) & protein (Western or by

some specific assay method)

Expression will vary in transgenic

offspring: due to position effect and

copy number


10/22

An organism that carries

a transgene in its germ line &

can be used in matings to

establish a pure breeding

transgenic line or one that act

as a breeding stock fortransgenic animals


11/22


12/22

The incorporation of injected DNA into the mouse genome cannot

be tightly controlled. As a result, improper incorporation may

occur.

If DNA is incorporated at the 2-cell stage, some cells may expressthe DNA while others will not (mosaic mice).

If the injected DNA is incorporated in multiple sites, multiple

copies of the DNA may be expressed leading to overexpression.

If the DNA copy is not incorporated into the germ line, then it will

not be passed to offspring.

Problems with Pronuclear Injection


13/22

Less than 5% of

the microinjected

fertilized eggs

become transgenic

progeny


14/22

Retroviral vectors can be used to

create transgenic animals

To increase the probability of expression, gene

transfer is mediated by means of a vector,

generally a virus.

Retroviruses are commonly used as vectors to

transfer genetic material into the cell, taking

advantage of their ability to infect host cells in

this way.

Offspring derived from this method arechimeric, i.e., not all cells carry the retrovirus.

Transmission of the transgene is possible only

if the retrovirus integrates into some of the

germ cells


15/22

Genetically engineered

embryonic stem (ES)

cells can be used to

create transgenic animals

but this method is labor

intensive & used to

allow for gene targetingvia homologous

recombination.


16/22

Engineered embryonic stem cellmethod

Step 1: Get the ES cells


17/22

Step 2: Genetically engineer the ES cells


18/22

Step 3:

Place engineered ES

cells into an earlyembryo


19/22

Transgenic mouse: Marathon Mice

The genetically enhanced

marathon mice (picture) ran

twice as far and nearly twice as

long as ordinary rodents. The

peroxisome proliferator activated

receptor (PPAR-delta) gene was

overexpressed in these transgenic

mice. The enhanced PPAR-delta

activity increased fat burning.


20/22

Cloning livestock by

nuclear transfer


21/22

Establishing transgenic chickens by

transfection of isolated blastoderm

cells Resistance to viral and bacterial

disease.

Better feed efficiency.

Lower fat and cholesterol levelsin eggs.

Better meat quality.

Eggs with pharmaceutical

proteins.


22/22

Some human proteins expressed in the mammary

glands of transgenic animals

Erythropoietin

Factor VIII & IX

Fibrinogen

Growth hormone

Hemoglobin Insulin

Monoclonal antibodies

Tissue plasminogen activator (TPA)

Antitrypsin

Antithrombin III (the first transgenic animal drug, ananticlotting protein, approved by the FDA in 2009)

Documents

Lec # 2. Animal Biotechnology