B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Production & Applications of Transgenic Animals:

Production of Pharmaceuticals, Production of donor organs, Knock out mice

Transgenic Animals:

A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its

genome. The foreign gene is constructed using recombinant DNA methodology. In addition to

the gene itself, the DNA usually includes other sequences to enable it

• to be incorporated into the DNA of the host and

• to be expressed correctly by the cells of the host.

• Transgenic sheep and goats have been produced that express foreign proteins in their

milk.

• Transgenic chickens are now able to synthesize human proteins in the "white" of their

eggs.

These animals should eventually prove to be valuable sources of proteins for human therapy.

In July 2000, researchers from the team that produced Dolly reported success in producing

transgenic lambs in which the transgene had been inserted at a specific site in the genome and

functioned well.

Transgenic mice have provided the tools for exploring many biological questions.

An example:

Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in

humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive,

easily-manipulated model for studying the disease. However, transgenic mice expressing the

human gene for the polio virus receptor

• can be infected by polio virus and even

• develop paralysis and other pathological changes characteristic of the disease in humans.

Production of Transgenic Animals

Two methods of producing transgenic mice are widely used:

• transforming Embryonic stem cells (ES cells) growing in tissue culture with the desired

DNA;

• injecting the desired gene into the Pronucleus of a fertilized mouse egg.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

1) The Embryonic Stem Cell Method (Method "1")

Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse

blastocysts. They can be grown in culture and retain their full potential to produce all the cells of

the mature animal, including its gametes.

Figure 1: Embryonic Stem Cell method

i) Make your DNA

Using recombinant DNA methods, build molecules of DNA containing

• the gene you desire (e.g., the insulin gene);

• vector DNA to enable the molecules to be inserted into host DNA molecules;

• promoter and enhancer sequences to enable the gene to be expressed by host cells.

ii) Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.

iii) Select for successfully transformed cells. [Method]

iv) Inject these cells into the inner cell mass (ICM) of mouse blastocysts.

v) Embryo transfer

• Prepare a pseudo-pregnant mouse (by mating a female mouse with

a vasectomized male). The stimulus of mating elicits the hormonal changes needed to

make her uterus receptive.

• Transfer the embryos into her uterus.

• Hope that they implant successfully and develop into healthy pups (no more than one-

third will).

vi) Test her offspring

• Remove a small piece of tissue from the tail and examine its DNA for the desired gene.

No more than 10–20% will have it, and they will be heterozygous for the gene.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

vii) Establish a transgenic strain

• Mate two heterozygous mice and screen their offspring for the 1 in 4 that will

be homozygous for the transgene.

• Mating these will found the transgenic strain.

2) The Pronucleus Method (Method "2")

i) Prepare your DNA as in Method 1

ii) Transform fertilized eggs

• Harvest freshly fertilized eggs before the sperm head has become a pronucleus.

• Inject the male pronucleus with your DNA.

• When the pronuclei have fused to form the diploid zygote nucleus, allow the

zygote to divide by mitosis to form a 2-cell embryo.

iii) Implant the embryos in a pseudo-pregnant foster mother and proceed as in Method 1.

An Example

Figure 2: Image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse

(right) with a normal littermate (left). The giant mouse developed from a fertilized egg

transformed with a recombinant DNA molecule containing:the gene for human growth

hormone and a strong mouse gene promoter

The levels of growth hormone in the serum of some of the transgenic mice were several

hundred times higher than in control mice.

Random vs. Targeted Gene Insertion

The early vectors used for gene insertion could, and did, place the gene (from one to 200 copies

of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a

particular gene, it is possible to design vectors that replace that gene. The replacement gene can

be one that restores function in a mutant animal or knocks out the function of a particular locus.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

In either case, targeted gene insertion requires the desired gene

• neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its

relatives, like the drug G418, which is lethal to mammalian cells;

• tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside

analogue ganciclovir. DNA polymerase fails to discriminate against the resulting

nucleotide and inserts this non-functional nucleotide into freshly-replicating DNA. So,

ganciclovir kills cells that contain the tk gene.

Figure 3: Random vs Targeted Gene Insertion

Step 1: Treat culture of ES cells with preparation of vector DNA.

Results:

• Most cells fail to take up the vector; these cells will be killed if exposed to G418.

• In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire

vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but

killed by gancyclovir.

• In still fewer cells: homologous recombination occurs. 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.

Step 2: Culture the mixture of cells in medium containing both G418 and ganciclovir.

• The cells (the majority) that failed to take up the vector are killed by G418.

• The cells in which the vector was inserted randomly are killed by gancyclovir (because they

contain the tk gene).

• This leaves a population of cells transformed by homologous recombination (enriched several

thousand fold).

Step 3: Inject these into the inner cell mass of mouse blastocysts.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Applications of Transgenic Animals

i) As disease model: Historically, mice have been used to model human disease because of their

physiological, anatomical and genomic similarities to humans. Transgenic animals are produced

as disease models (animals genetically manipulated to exhibit disease symptoms so that effective

treatment can be studied) such as Alzheimers, cancer, AIDS. Transgenic animals enable scientists

to understand the role of genes in specific diseases. Advantage of using transgenic animals is the

replacement of higher species by lower species- through development of disease models in mice

rather than in dogs or non-human primates, the extent of discomfort experienced by parent

animals during the experimental procedures. Transgenic animals such as mice have been found

to be valuable in investigations into gene function and for analysis of different hereditary

diseases.

ii) As food: The FDA suggested that cloned animals and their products were safe to eat for human

being. Some drawbacks are associated due to their muscle hypertrophy like difficulties in calving

requiring Caesareans, poor viability of calves and poor fertility.

iii) Drug and Industrial production: Transgenic animals are used for production of proteins

such as alpha-1-antitrypsin, produced in liver, used in treatment of emphysema or cystic fibrosis.

Less expensive than production of protein through culture of human cells.

The human lungs are constantly get affected by foreign particles such as dust, spores and bacteria.

To prevent these, neutrophils releasing the elastase enzyme but this enzyme harmed the elastin

in the lungs which maintains the elasticity of lungs. So, human body releases a protein α1

proteinase inhibitor which has been successfully expressed in sheep. Recombinant human

proteins produced in the mammary glands of transgenic animals. Pharmaceutical proteins are

now used for commercial purpose. Two scientists at Nexia Biotechnologies in Canada spliced

spider genes into the cells of lactating goats. The goats are used to manufacture silk, milk and

secrete tiny strands from their body by the bucketful. By extracting polymer strands from the

milk and weaving them into thread which is light and tough material that could be used to prepare

military uniforms, medical micro sutures and tennis racket strings. Americans are more

supportive (60%) for above use of transgenic animals. The mammary gland of transgenic goats

is used to produce Monoclonal Antibodies. A recombinant bispecific antibody is produced by

using transgenic cattle with in their blood.

Another application includes newly generation of trans-chromosomal animals in which a human

artificial chromosome containing the complete sequences of the human immunoglobulin heavy

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

and light chain loci was introduced into bovine fibroblasts, which were then used in nuclear

transfer. Transchromosomal bovine offspring were obtained that expressed human

immunoglobulin in their blood. This could be a significant step forward in the generation of

human therapeutic polyclonal antibodies.

iv) Disease control: Scientist developed the mice by altering the genes of the mousepox virus in

Australia. Some scientist also thought to develop genetically modify mosquitoes so they cannot

produce malaria but other scientist worry about these mosquitoes that they could have unforeseen

possibly risk if, they are released into the environment.

v) Xenotransplantation: Now a day approximately about 250000 people are alive due to the

successful transplantation of an appropriate allotransplantation. Sometimes there is limitation of

appropriate organs or rejection of live organ donation. So, to rectify this problem porcine

xenografts from domesticated pigs are considered to be the best choice. Pigs which are

genetically modified can be used as a source animal for tissues and organs in human beings for

transplantation purpose by delete the gene responsible for the human rapid immune rejection

response. In Canada, a National survey on xenotransplantation showed that only 48% found

acceptable for ‘the use of animals as a source of living cells, tissues or organs to prolong human

life. To overcome the Hyperacute rejection & acute vascular rejection, synthesis of human

regulators of complement activity are produced in transgenic pigs. Survival rates, after the

transplantation of porcine hearts or kidneys expressing transgenic regulators of complement

activity proteins to immunosuppressed nonhuman primates, reached near about 23 to 135 days.

So, the Hyperacute rejection can be overcome in a clinically acceptable manner. For long term

graft tolerance induction of permanent chimerism via intraportal injection of embryonic stem

(ES) cells or the co-transplantation of vascularised thymic tissue.

vi) Blood replacement Transgenic swine are used to produce human haemoglobin. The protein

obtained from transgenesis could be purified by using procine blood which is similar to human

haemoglobin.

vii) Agriculture Transgenic pigs containing a human metallothionein promoter or porcine

growth-hormone gene construct referred significant improvements in economically traits

including growth rate, body fat/muscle ratio. Transgenic pigs are used to produce pork by using

spinach desaturase gene which produce large amount of non-saturated fatty acids, used for diet

purpose and was advantageous to reduce the risk of stroke and coronary disease. Transgenic

animals are used for milk production. Generally, there is an improvement in milk composition.

For this purpose transgenic mice have been developed, but adverse effects are often seen.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Transgenic pigs are used to increase milk production by altering the composition of lactose. In

the pig, transgenic expression of a bovine lactalbumin construct in sow milk has been resulting

in higher lactose contents and greater milk yields, correlated with improved survival and

development of piglets 68. Transgenic sheep are used for wool production in which transgenic

sheep carrying a keratin-IGF-I construct showed that expression in the skin and the amount of

clear fleece was about 6.2% greater in transgenic as compared to nontransgenic animals 69, 70.

Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs,

but a very limited number of genes are currently known to be responsible for resistance to

diseases in farm animals 71.

viii) Transgenic animals are used in toxicity testing.

ix) Transgenic animals are used for vaccine testing.

Source: https://ijpsr.com/bft-article/transgenic-animals-production-and-

application/?view=fulltext

Production of Pharmaceuticals

A new brand of farming is emerging from the research and development labs of several

universities and small biotechnology companies-so new they're even changing the spelling to

"pharming."

Pharming is the production of human pharmaceuticals in farm animals that is presently in the

development stage with possible commercialization by the year 2000. It has been gaining

application among biotechnologists since the development of transgenic "super mice" in 1982

and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator

to treat blood clots), in 1987. Transgenic organisms have been modified by genetic engineering

to contain DNA from an external source. The first drugs produced by this approach are about to

enter clinical trials as part of the FDA review process. These transgenic animals will likely be

raised by the pharmaceutical companies and will certainly be kept separate from the food supply.

Many pharmaceutical drugs in the form of complex proteins require 3D structures that are

important for their functions. Animal cells have the unique machinery to make the special

structures. Genetically engineered (transgenic, GMO) animals/animal cells are created so they

serve as “bioreactors” to produce these drugs at an industrial scale. Animal products such as milk,

egg white, blood, urine, and silk worm cocoons have been used to produce complex drugs that

can’t be made by chemical synthesis. The first drug produced by GMO animals, anti-thrombin

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

III from the milk of transgenic goats, prevents the formation of small blood clots that could break

loose and plug other vessels (Figure 1).

Figure: Generation of human anti-thrombin III by transgenic goats

Source: https://gmo.uconn.edu/topics/pharmaceutical-use-of-gmos/#

http://www.biotech.iastate.edu/publications/biotech_info_series/bio10.html

It was approved by the FDA in 2009. Animal cells and simple bacteria, however, have been used

to produce protein drugs much earlier than that. For example, Activase® (r-tPA), produced by

cells from Chinese hamsters, was approved by the FDA to treat stroke since 2001. The first

bacteria produced drug, Humulin (human insulin) from Eli Lilly has been used by millions if not

billions since 1982. Today, many cancer drugs such as monoclonal antibody therapeutics are

produced by animal cell cultures after human genes are introduced to these cells. Pharmaceuticals

from GMO animals/GMO cells/GMO bacteria will continue to be developed to save lives.

Although most protein drugs are made in milk, a notable exception is human hemoglobin that is

being made in swine blood to provide a blood substitute for human transfusions. Because

hemoglobin is naturally a blood protein, it is likely to be one of few exceptions to the usual

method of production in milk. Furthermore, the economics of blood production are less

favorable, because to recover human hemoglobin, the animal producing it must be slaughtered.

Drugs currently made by or being developed in transgenic animals are listed in Table 1. Notice

that pharming is expected to increase the value of animals dramatically. In general, animal

pharming is considered to be 5 to 10 times more economical on a continuing basis and 2 to 3

times cheaper in startup costs than cell culture production methods.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Figure : Biologics from Transgenic animal milk promise treatments for many ailments

Source: https://cen.acs.org/articles/93/i19/Producing-Protein-Therapeutics-Water-

Buffalo.html

Regulatory and Ethical Issues

Production of human pharmaceuticals in farm animals has many technical barriers to overcome,

although most technologists agree that these technical difficulties will be easily resolved in the

1990s. As a production method, animal pharming is entirely unprecedented and is likely to

undergo significant evaluation by the Food and Drug Administration (FDA). Human drugs

purified from animal milk or blood are likely to require exceptional levels of safety testing before

animal and human health concerns are addressed to the satisfaction of consumers. At a more

fundamental level, many people are genuinely concerned about animal welfare and

biotechnology's redefinition of the relationship between humans and animals. Genetic

engineering and transgenic animal research are essentially human endeavors to improve the

availability, quality, and safety of drugs; to enhance human health; and to improve animal health.

Animal breeding has gone on for centuries, but the ability to change the DNA of the animal

brings breeding to a revolutionary new level.

Pharmaceutical proteins: Some samples of biomedicines recently expressed in plants

include erythropoietin, interferon, hirudin, aprotinin, Leu-enkephalin, somatotropin of human

growth hormone

Non-pharmaceutical proteins derived from plants or industrial proteins belong mainly to the

enzymes that include avidin, trypsin, aprotinin, β-glucocerebrosidase,

peroxidase and cellulose, etc., listed by Basaran and Rodriguez-Cerezo and now available in the

market. Molecular farming of destructive enzymes of the cell walls such as

cellulose, hemicellulase, xylanase, and particularly ligninase provide a great status for the

biofuel industry respecting cellulosic ethanol

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Molecular Farming and metabolic engineering, an opportunity for producing plants with

a high technology

Molecular farming and metabolic engineering make the production of new high-tech products

possible. There is a driving force backing molecular farming that makes its costs much less than

traditional farming. Chlamydomonas reinhardtii, as a unicellular alga, is one of the most recent

production projects examined by Franklin and Mayfield. C. Reinhardtii is the only plant whose

transformation was operated in its all segments containing DNA (nucleus, plastid, and

mitochondria). Unique features of the moss system bring about the possibility of removing target

genes and purification of the proteins secreted from the culture medium. The target gene was

omitted to get rid of the nuclear genes for glycosylation. The first step towards the long-term

goals of reengineering mechanism in modifications of plant proteins is setting a new standard in

all systems of plant expressions in order to humanize the biomedicines produced in plants. The

aim of molecular farming is to produce large quantities of active and secure pharmaceutical

proteins with lower prices.

Production of Donor Organs

Organ transplantation is a treatment option for people who have end-stage organ failure that can’t

be controlled using other treatments.

Organ transplantation is one of the greatest technological achievements of modern medicine, but

the ability of patients to benefit from transplantation is limited by shortages of transplantable

organs.

Regenerative medicine, including cell transplantation therapy and tissue engineering, offers an

effective approach for developing treatments of intractable diseases. Still, organ transplantation

has been the last line of therapy in saving patients experiencing end-stage organ failure when

othe rtherapies are not effective. However, the growing shortage of organ donors throughout the

world is still a major challenge.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Figure : Schematic presentation of blastocyst complementation: generation of human

organs using organogenesis-disabled pigs as a platform. iPS cells, inducedpluripotent stem

cells. [H. Nagashima, H. Matsunari / Theriogenology 86 (2016) 422–426423]

Source:https://reader.elsevier.com/reader/sd/pii/S0093691X16300954?token=405F5F4D7

D17804B0B596097FAD41C69546149E708EE44BD437783042C62A5F0B3AEABF26D5E

AAD4D1AEA6F1427D28EF

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Figure 2: The strategy of tolerance via bone marrow transplantation. Before

transplantation, the recipient undergoes pre-conditioning to make room for the incoming

donor bone marrow cells. An organ is transplanted at the same time as donor bone marrow.

At first, the recipient is put on immunosuppressive drugs so that the donor bone marrow

cells don’t get rejected. Donor immune cells are monitored for signs of immunoreactivity

against the transplanted organ. Once these tests show no immunoreactivity, the recipient

can reduce the amount of immunospressive drugs that they are taking. Organ function is

monitored until complete medication withdrawal of is achieved.

Source:http://sitn.hms.harvard.edu/flash/2015/xenotransplantation-can-pigs-save-human-

lives/

Source: https://www.straitstimes.com/world/united-states/scientists-trying-to-grow-

human-organ-in-human-pig-embryo

The concept of cross-species (pig-to-human) transplantation is known as xenotransplantation,

and the transplanted organs or tissues are called xenografts. The first xenograft heart transplant

in a human was performed in 1964, using a chimpanzee heart. Thomas Starzl carried out the first

chimpanzee-to-human liver transplantation in 1966, and in 1992, he performed a baboon-to-

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

human liver transplant. In each of these cases, the survival of the xenografts was short due to

graft rejection. Immunosuppressive drugs were not enough to prevent an immune response

against the xenografts.

Chimeras

Named for the mythological composite beast, a chimera--broadly defined--can be a composite of

two individuals (or even species) at any level

• organism - embryonic cells from one individual (donor) are transplanted into the embryo of

another individual (recipient), resulting in a chimeric recipient

• cell - a cell containing chimeric DNA will express traits encoded by both donor species' genes

• nucleic acid - DNA or RNA spliced together from two different individuals

Source: https://www.dailynews.com/2015/11/07/us-officials-promise-to-create-new-policies-for-

suspended-chimera-research/

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

Figure : Human ear growing in nude mice

http://havacuppahemlock1.blogspot.com/2016/08/human-animal-chimera-models-creating.html

Knockout Mice:

If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the

heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the non-

functional gene (both copies of the gene at that locus have been "knocked out").

Knockout mice are valuable tools for discovering the function(s) of genes for which mutant

strains were not previously available. Two generalizations have emerged from examining

knockout mice:

• Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out

not to be indispensable. The mouse genome appears to have sufficient redundancy to

compensate for a single missing pair of alleles.

• Most genes are pleiotropic. They are expressed in different tissues in different ways and

at different times in development.

Tissue-Specific Knockout Mice

While "housekeeping" genes are expressed in all types of cells at all stages of development, other

genes are normally expressed in only certain types of cells when turned on by the appropriate

signals (e.g. the arrival of a hormone).

To study such genes, one might expect that the methods described above would work.

However, it turns out that genes that are only expressed in certain adult tissues may nonetheless

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

be vital during embryonic development. In such cases, the animals do not survive long enough

for their knockout gene to be studied.

Fortunately, there are now techniques with which transgenic mice can be made where a

particular gene gets knocked out in only one type of cell.

The Cre/loxP System

One of the bacteriophages that infects E. coli, called P1, produces an enzyme - designated Cre

— that cuts its DNA into lengths suitable for packaging into fresh virus particles. Cre cuts the

viral DNA wherever it encounters a pair of sequences designated loxP. All the DNA between

the two loxP sites is removed, and the remaining DNA ligated together again (so the enzyme is

a recombinase).

Using "Method 1" (above), mice can be made transgenic for

• the gene encoding Cre attached to a promoter that will be activated only when it is

bound by the same transcription factors that turn on the other genes required for the

unique function(s) of that type of cell;

• a "target" gene, the one whose function is to be studied, flanked by loxP sequences.

Figure 4: Production of Knock out mice: The Cre/loxP System

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

In the adult animal,

• those cells that

o receive signals (e.g., the arrival of a hormone or cytokine)

o to turn on production of the transcription factors needed

o to activate the promoters of the genes whose products are needed by that

particular kind of cell

will also turn on transcription of the Cre gene. Its protein will then remove the "target"

gene under study.

• All other cells will lack the transcription factors needed to bind to the Cre promoter

(and/or any enhancers) so the target gene remains intact.

The result: a mouse with a particular gene knocked out in only certain cells.

Knock-in Mice

The Cre/loxP system can also be used to

• remove DNA sequences that block gene transcription. The "target" gene can then be

turned on in certain cells or at certain times as the experimenter wishes.

• replace one of the mouse's own genes with a new gene that the investigator wishes to

study.

Such transgenic mice are called "knock-in" mice.

Transgenic Sheep and Goats

Until recently, the transgenes introduced into sheep inserted randomly in the genome and often

worked poorly. However, in July 2000, success at inserting a transgene into a specific gene

locus was reported. The gene was the human gene for alpha1-antitrypsin, and two of the

animals expressed large quantities of the human protein in their milk.

This is how it was done.

Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with

a vector that contained these segments of DNA:

1. 2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen.

(Its absence in humans causes the inherited disease osteogenesis imperfecta.)

This locus was chosen because fibroblasts secrete large amounts of collagen and thus

one would expect the gene to be easily accessible in the chromatin.

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

2. A neomycin-resistance gene to aid in isolating those cells that successfully incorporated

the vector. [Link to technique]

3. The human gene encoding alpha1-antitrypsin.

Some people inherit two non- or poorly-functioning genes for this protein. Its resulting

low level or absence produces the disease Alpha1-Antitrypsin

Deficiency (A1AD or Alpha1). The main symptoms are damage to the lungs (and

sometimes to the liver).

4. Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene

expression in milk-producing cells.

5. Binding sites for ribosomes for efficient translation of the beta-lactoglobulin mRNAs.

Successfully-transformed cells were then

• fused with enucleated sheep eggs [Link to description of the method] and

• implanted in the uterus of a ewe (female sheep).

• Several embryos survived until their birth, and two young lambs lived over a year.

• When treated with hormones, these two lambs secreted milk containing large amounts

of alpha1-antitrypsin (650 µg/ml; 50 times higher than previous results using random

insertion of the transgene).

On June 18, 2003, the company doing this work abandoned it because of the great expense of

building a facility for purifying the protein from sheep's milk. Purification is important because

even when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep

proteins that remain.

However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won

preliminary approval to market a human protein, antithrombin, in Europe. Their protein

- the first made in a transgenic animal to receive regulatory approval for human therapy

- was secreted in the milk of transgenic goats.

Transgenic Chickens

Chickens

• grow faster than sheep and goats and large numbers can be grown in close quarters;

• synthesize several grams of protein in the "white" of their eggs.

Two methods have succeeded in producing chickens carrying and expressing foreign genes.

• Infecting embryos with a viral vector carrying

o the human gene for a therapeutic protein

B.Sc. (Hons) Zoology Dr Anita K. Verma

Biotechnology Associate Professor

Sem VI Kirori Mal College

o promoter sequences that will respond to the signals for making proteins

(e.g. lysozyme) in egg white.

• Transforming rooster sperm with a human gene and the appropriate promoters and

checking for any transgenic offspring.

Preliminary results from both methods indicate that it may be possible for chickens to produce

as much as 0.1 g of human protein in each egg that they lay.

Not only should this cost less than producing therapeutic proteins in culture vessels, but

chickens will probably add the correct sugars to glycosylated proteins — something that E.

coli cannot do.

Transgenic Pigs

Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have

incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may

someday be able to produce transgenic pigs that can serve as a source of transplanted organs for

humans.

Transgenic Primates

In the 28 May 2009 issue of Nature, Japanese scientists reported success in creating transgenic

marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically

engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into

the germline and passed on to the animal's offspring. The hope is that these transgenic animals

will provide the best model yet for studying human disease and possible therapies.

References

Frank Gwazdauskas, professor, dairy science department, Virginia Polytechnic Institute and

State University, Blacksburg, Virginia 24061-0315.

"See How They (Don't) Grow." Successful Farming. March 1991, p. 33.

"Transgenic Animals in the Production of Therapeutic Proteins." Biotechnology International.

Century Press, 1992, p. 317.

"Transgenic Pharming Advances." Bio/Technology. May 1992, p. 498.

"Whole Animals for Wholesale Protein Production." Bio/Technology. August 1992, p. 863.

Table 1