Limitations of Using Transgenic Goats to Produce Spider Silk

Original paragraph

While making larger quantities of artificial spider silk is already a reality, getting it to a commercially viable level, at which vast amounts can be consistently produced, is not.

The difficulty lies in finding the right gene to code for the right type of silk protein (each spider makes several silks, with different tensile and elastic properties) and in finding the most efficient way to get other organisms to make a lot of it, whether goats, plants or tankfuls of E. coli.

Source
Torres, Phil. "Spiders and Transgenic Goats Lead to New €˜silk Road | Al Jazeera America." Spiders and Transgenic Goats Lead to New €˜silk Road | Al Jazeera America. N.p., 2 May 2014. Web. 17 Oct. 2014. <http://america.aljazeera.com/watch/shows/techknow/articles/2014/5/1/spiders-and-transgenicgoatsleadtonewasilkroada.html>.

Original paragraph

The process of transgenic production is only 10-25% efficient in mice and 1-5% in larger mammals. We have discussed the problems of DNA transfer, but we also need to consider the process of IVF itself. IVF is relatively efficient in mice, but not so in larger mammals and in humans the success rate is about 20%. . Current work suggests that it is not a problem with the technique but that larger animals are inherently less efficient with respect to implantation of the embryos with large numbers wasted. Additionally, insertional mutagenesis will kill or disable some of the pups. A number of transgenics are infertile and these will be useless for future breeding programmes, although if cloning becomes more efficient we may be able to propagate these animals. The sex of the embryo is also important in recombinant protein production and foetal sex analysis has been used in the past allowing early abortion of male transgenics, pre-implantation DNA analysis allows sexing of the eggs prior to transfer.

The time and effort involved in producing useful transgenic animals is immense. The larger animals have long gestation periods and mature slowly. Mice provide less of a problem in this respect. It is also financially draining to keep animals that may not be useful. Additionally, if only 1% of the animals are transgenic all the others must be slaughtered. Since they are genetically modified they cannot be used as foodstuff and must be incinerated. Clearly there is scope for the introduction of new more efficient methods that will allow us to be sure that each experiment yields a transgenic animal with the appropriate gene incorporated at a single chosen site in the genome.

PROBLEMS

efficiency - in mice is only 10-25% in terms of DNA incorporation, in cattle, sheep and goats is 1-5%

insertional mutagenesis kills some of the pups

some of the transgenic pups are infertile

they may not be of the right sex for the purpose e.g. milk production

the time and effort involved are huge

in larger animals the gestation times are long and offspring are not sexually mature for a year

if 1/100 sheep born are transgenic the rest will be slaughtered and the carcasses burnt

larger animals only produce 1 or 2 offspring

viruses/bacteria etc

Source

"Transgenic Animals." Journal of Equine Veterinary Science 22.2 (2002): 94. Web. 18 Oct. 2014. <http://www2.wmin.ac.uk/~redwayk/pgcert_DL/molecular_therapeutics/files/Chapter%206.pdf>.

Original paragraph

During a goat transgenic program that took place in Israel from July 1995 to February 1996, Saanen (n = 343) and Nubian x Damascus (n = 378) crossbred goats of mixed ages were used as donors (n = 433) and recipients (n = 288). The effects of season, age, number of surgical procedures, previous hormonal treatments and ovulation rate on the number of microinjectable embryos collected were studied. Likewise, the effects of these parameters on the pregnancy rate as well as the number of embryos transplanted, endogenous progesterone concentrations and exogenous progesterone supplementation were studied in recipient does. Following superovulation with ovine follicle stimulating hormone, 85% of the does responded with 13.6 +/- 5.7 (mean +/- S D) ovulations/doe. Age, month and number of previous hormonal treatments significantly affected the ovulation rate. The average recovery rate was 70%, and it was affected only by the ovulation rate. Pronuclei were visualized in about 30% of the flushed embryos (including unfertilized ova), and those were microinjected with human serum albumin gene construct. About 68% of the injected embryos underwent at least one division during an overnight incubation, and those embryos were transferred, giving about 2.0 transferred embryos per ovulated donor. Of the recipients, 86% responded following synchronization with 3.1 +/- 1.6 (mean +/- S D) ovulations per doe. Breed and month had a significant effect on the ovulation rate. Two or three microinjected embryos were transferred to each recipient, resulting in more than a 40% pregnancy rate during September to November. Lower pregnancy rates were obtained before and after that period. By monitoring plasma progesterone concentrations in the recipients it was found that progesterone concentration was correlated with the ovulation rate. However, the pregnancy rate was not affected by progesterone concentration. During January and February, 30 to 50% of the recipients failed to develop functional corpora lutea (CL) following embryo transfer, which explained the lower pregnancy rate in those months. Of the 86 kids born 4 were transgenic.

Source
Goodwine, E. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 18 Oct. 2014. <http://www.ncbi.nlm.nih.gov/pubmed/16728144>.

Key ideas:

• The difficulty is finding the right gene code in a spider for its dragline silk
• Also finding the most efficient way to make other organisms to produce it
• The offspring goat might not be the right sex for milk production
• The success rate is small - only about 5%

Paraphrasing

There are several limitations of using transgenic goats to produce spider silk. Among them are the difficulty in finding the gene codes and the host organism, several problems that can arise in the offspring and the very small success rate of the process. One of the difficulties lies in finding the right gene code in a spider for its dragline silk. A single spider produces a variety of silk types and finding the right gene that codes for the strongest and most elastic silk is difficult. In addition to that, scientists still do not know what is the most efficient way to make other organisms produce silk. Goats might be the most effective solution to this problem. The next limitation is problems that can arise in the offspring. One of these problems is that the goat produced with the spider transgene might not be right sex for milk production. This is crucial because scientists rely on the milk production as a natural outlet for the silk. If the goat is male, it would be harder to milk than if it were female. The last limitation is the overall small success rate. The results of a transgenic goat program conducted in Israel from July 1995 to February 1996 is that of the 86 kids born, only 4 were transgenic. This gives us about a 5% success rate of an offspring goat having the spider transgene in them. These limitations are the reasons as to why there are not more companies using transgenic goats to produce spider silk. Especially with the example of Nexia Biotechnologies which infamously went bankrupt after successfully using transgenic goats to produce spider silk.

Benefits of Using Transgenic Goats to Produce Spider Silk

Original paragraph

Here’s a breakdown of a few of the industries in which spider silk can be used:

Medicine As far as researchers can tell, spider silk is all but invisible to the human body, meaning that our immune systems won’t reject it as they occasionally do with other substances. This makes the elastic and tensile properties of spider silk perfect for making artificial tendons, cartilage and ligaments as well as sutures and sealants. Prototypes of these already exist.

Military As we saw while shooting this story, a layered piece of rubbery silicone with 10 layers of spider silk painted on was enough to stop a bullet. Without those incredibly thin layers of spider silk, the bullet went right through. Properties like this make it an excellent candidate for body and vehicle armor.

Sports Spider silk proteins, when painted as a sheet, have incredibly absorbent qualities for dissipating impact and shock, like from bullets. This could translate to football helmets that protect our heads better than any before.

Automotive Tires and airbags are standard in every vehicle and could soon incorporate spider silk protein to make them stronger and lighter than ever.

Source
Torres, Phil. "Spiders and Transgenic Goats Lead to New €˜silk Road | Al Jazeera America." Spiders and Transgenic Goats Lead to New €˜silk Road | Al Jazeera America. N.p., 2 May 2014. Web. 17 Oct. 2014. <http://america.aljazeera.com/watch/shows/techknow/articles/2014/5/1/spiders-and-transgenicgoatsleadtonewasilkroada.html>.

Original paragraph

Scientists like working with goats for several reasons; one imagines their tendency not to eat one another comes in near the top of the list. They’re also easy to breed and handle, and pound-for-pound, goats produce as much milk as cows. But while a goat doesn’t weigh any more than a person, a cow weighs half as much as a car.

Source
Ferguson, Cat. "Mixing Spider DNA and Goat Embryos Produces Milk With a Side of Silk." TakePart. N.p., n.d. Web. 09 Oct. 2014. <http://www.takepart.com/article/2014/04/10/gmo-goats>.

Key ideas:

• Spider silk can produce materials like never before in multiple industries including medicine, military, sports and automotive
• Goats are farm friendly animals
• Goats are easy to breed and handle
• Goats produce as much milk as cows pound-for-pound

Paraphrasing

There are three benefits to using transgenic goats to produce spider silk. The first one is that just by using the transgenic goats, stronger and more elastic materials than ever before can be produced. These materials include those in the industries of medicine, military, sports and automotive. The second benefit is that goats are farm friendly animals. They can be kept in large numbers together because they are not cannibalistic like the spiders. In addition to that, they are also easy to breed and handle. The last and most important benefit of using transgenic goats is that they, pound-for-pound, produce as much milk as cows. A cow weighs about half of a car while a goat does not weigh anymore than an average person. These reasons are why the scientists chose the goats to become the transgenic species in the first place.

DNA Microinjection

Original paragraph

Generation of transgenic mice is possible because introduced DNA integrates into the germ line. The foreign DNA can then direct transcription in a predictable manner, and the genetic manipulation is transmitted to future generations (due to integration into the germ line), thus establishing a new and novel strain of the organism. First reported decades ago by Gordon and coworkers, the fundamental principles remain largely true to this day (Gordon and Ruddle, 1981). The procedure consists of preparation of the transgene, embryo collection, microinjection, transfer of the embryos, and identification of the transgenic offspring; these steps have been described more recently by Nagy and coworkers in great detail.(Nagy et al., 2003).

Key ideas:

• Transgenic animals are possible through DNA microinjection because introduced DNA from other species integrates into the germ line
• The foreign DNA can direct transcription in a predictable manner and the genetic manipulation is passed down to future generations
• The fundamental principles used in 1981 are still used today with no major changes
• The procedure consists of the preparation of the transgene, the embryo collection, the microinjection, the transfer of the embryos and the identification of the transgenic offspring

Source 

DeMayo, Janet L., Jie Wang, Dongcai Liang, Ruina Zhang, and Francesco J. DeMayo. "Genetically Engineered Mice by Pronuclear DNA Microinjection." N.p., n.d. Web. 15 Oct. 2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459342/>.

Paraphrasing

DNA microinjection is the method used to make transgenic goats that produce spider silk. This technique successfully produces transgenic goats because introduced DNA from the spider integrates into the germs line and is passed along to every cell in the goat's body. The principles first used in 1981 are still used today with no major changes. The procedure for DNA microinjection consists of five basic protocols which are the preparation of spider transgene DNA for microinjection, embryo collection from donor goats, DNA microinjection into the goat embryos, embryo transfer into female goats and identification of transgenic goats. Figure 2 below is an illustration of the DNA microinjection itself, the third basic protocol.

Figure 2: An illustration of DNA microinjection
http://images.tutorvista.com/content/biotechnology/micro-injection-process.jpeg

In the preparation of the spider transgene DNA for microinjection, the part of the genes of the spider that codes for the production of its dragline silk is removed from one of its cells and then prepared for microinjection. In the embryo collection from the donor goats, there are two options - either to let the female egg fertilize naturally or use in vitro fertilization (IVF). For the natural process, the scientists have to find a healthy female goat and a healthy male goat. The male goat would then be allowed to fertilize the egg of the female goat naturally. Then the scientists extract what now is an embryo. For the IVF process, one healthy egg is chosen from the female goat and sperms from the male goat are placed in a petri dish. The fertilization will occur here thus forming an embryo. In the DNA microinjection, the transgene from the spider is injected into the extracted goat embryo using a needle. The process under a microscope can be seen in video 2 below.

Video 2: DNA microinjection under a microscope

https://www.youtube.com/watch?v=h-Bfc1GPWpE

In the embryo transfer to female goats, the embryo containing the spider transgene is placed back inside a womb of a female goat and is then allowed to develop normally. The female goat will eventually give birth but the phenotype of the offspring will look just like normal. Therefore, to find out whether or not the DNA microinjection was successful, scientists will have to do the genetic testing. In the identification of the transgenic goats, scientists can extract any cell from the offspring and then completely analyze the DNA to see if the spider transgene is actually there. If it is there, it means that the DNA microinjection process was successful and that they have successfully created a goat that is able to produce spider silk.

How Are Transgenic Animals Produced?

Original paragraph

Since the discovery of the molecular structure of DNA by Watson and Crick in 1953, molecular biology research has gained momentum. Molecular biology technology combines techniques and expertise from biochemistry, genetics, cell biology, developmental biology, and microbiology.

Scientists can now produce transgenic animals because, since Watson and Crick’s discovery, there have been breakthroughs in:

The insertion of a foreign gene (transgene) into an animal is successful only if the gene is inherited by offspring.

The success rate for transgenesis is very low and successful transgenic animals need to be cloned or mated.

• recombinant DNA (artificially-produced DNA)
• genetic cloning
• analysis of gene expression (the process by which a gene gives rise to a protein)
• genomic mapping

The underlying principle in the production of transgenic animals is the introduction of a foreign gene or genes into an animal (the inserted genes are called transgenes). The foreign genes “must be transmitted through the germ line, so that every cell, including germ cells, of the animal contain the same modified genetic material.” (Germ cells are cells whose function is to transmit genes to an organism’s offspring.)

To date, there are three basic methods of producing transgenic animals:

• DNA microinjection
• Retrovirus-mediated gene transfer
• Embryonic stem cell-mediated gene transfer

Key ideas:

• Scientists now can produce transgenic animals because of several breakthroughs in recombinant DNA, genetic cloning, analysis of gene expression and genomic mapping
• The principle of the production of transgenic animals is the introduction of a foreign gene into an animal
• There are three methods of producing transgenic animals which are DNA microinjection, retrovirus-mediated gene transfer and embryonic stem cell-mediated gene transfer

Source 
Margawati, Endang T. "Transgenic Animals: Their Benefits To Human Welfare." Actionbioscience. N.p., n.d. Web. 06 Oct. 2014. <http://www.actionbioscience.org/biotechnology/margawati.html>.


Paraphrasing

Transgenic animals are able to be produced by scientists today because of several breakthroughs in recombinant DNA or artificially-produced DNA, genetic cloning, analysis of gene expression and genomic mapping.

The production of transgenic animals is basically the introduction of a foreign gene from another species into an animal. So far, there are three methods of producing transgenic animals which are DNA microinjection, retrovirus-mediated gene transfer and embryonic stem cell-mediated transfer.

Transgenic Goats

Original paragraph

The animals look and act just like regular goats: Workers milk them like they would at any dairy, and the goats raid their handlers’ pockets for stuff to chew on—a risk you face around any kid, doe, or billy goat. But scientists have added an extra protein or two to their already nutrient-rich milk.

One of those proteins is from the golden orb-weaver spider. Its silk, tougher than Kevlar, is a hundred times stronger than human ligaments. The potential for a light, incredibly resilient fiber is immense: The future might hold anything from spider knee replacements to spider parachutes. But aside from being viscerally disgusting, a spider farm just won’t work. Golden orb weavers are cannibalistic. In 2000, Nexia Biotechnology figured out a solution to that problem that reads like science fiction—it bred goats that can produce the silk instead.

To make the goats, scientists ferreted out the chunk of the golden orb weaver’s genetic code that’s responsible for directing the production of the single protein that makes up its dragline silk, a kind of safety line that connects the spider to its web. Next they tucked that gene into the DNA of goat embryos, alongside the natural milk production genes, to take advantage of the ready-made machinery. The embryos were then implanted into female goats, which gave birth to kids with a little something extra. Now, keeping the line going is easy—just breed a spider-goat with a normal one; half of the babies will carry the spider gene.

The only way to find out if they have the gene is by genetic testing. Lewis has several sets of fraternal twins where only one is part spider. He often asks visitors to guess which makes silk. “Nobody’s better than fifty-fifty guessing which is which,” he said.

Like any other goats, the transgenic ones mature around 18 months. That’s when they start lactating, and the spider silk proteins are mixed in with the milk—which Lewis and his crew extract from the goats’ udders with good old-fashioned milking. “All they want you to do is scratch their head,” Lewis said. “And to milk them, you just turn ’em loose, and they hop up to get milked.”

While getting the silk out is trickier than pouring milk into a bottle, it’s not fundamentally more complicated than making cheese. First the fat is skimmed off, and then the silk proteins are processed out, like separating curds from whey.

Key ideas:

• Scientists added protein from the golden orb-weaver spider into goats' milk
• Scientists found out which of the spider's genetic code is responsible for the production of the single protein that makes up its dragline silk
• They put that gene into the DNA of goat embryos next to the natural milk production genes
• The embryos were implanted into female goats
• The only way to find out if a goat has a gene or not is by genetic testing
• The goats mature after 18 months and then they start lactating
• To get silk out of the milk, first the fat is skimmed off and then the silk proteins are processed out

Source 
Ferguson, Cat. "Mixing Spider DNA and Goat Embryos Produces Milk With a Side of Silk." TakePart. N.p., n.d. Web. 09 Oct. 2014. <http://www.takepart.com/article/2014/04/10/gmo-goats>.
O'Brien, Miles, and Marsha Walton. "Research Areas." Nsf.gov. N.p., 03 May 2010. Web. 05 Oct. 2014. <http://www.nsf.gov/news/special_reports/science_nation/spidersilk.jsp>. 

Paraphrasing

The goats may look like any other but they are not. They are transgenic and have spider DNA in them. Scientists have added protein from the golden orb-weaver spider into the goats' milk in order to make it easier to harvest spider silk. First, the scientists found out which of the spider's genetic code is responsible for the production of the single protein that makes up its dragline silk. Next, they put that gene into the DNA of goat embryos next to the natural milk production genes. Those embryos were then implanted into female goats.

The only way to find out whether or not a goat has the spider gene is through genetic testing. Otherwise, they look just the same. Like normal goats, transgenic ones mature after around 18 months which is also when they start lactating. The spider silk proteins are in the goats' milk so to get them out you just have to milk the goats. However, getting the silk out of the milk is a bit harder. First, the fat is skimmed off and then the spider silk proteins are processed out. The video below from the National Science Foundation explains the things above further.

Video 1: All about transgenic goats

https://www.youtube.com/watch?v=ktgACq4zcAU

Why Spider Silk?

Original paragraph

Humans love spider webs, but aren't so crazy about their builders.

While spiders make some people flinch, there's no escaping the appreciation for their masterful web construction.

"There's a lot of interest in spider silk fibers because they're stronger than almost any other manmade fiber and they're also elastic," says Randy Lewis, professor of molecular biology at the University of Wyoming in Laramie.

Since ancient times, there's been a fascination with spider webs because of that combination of qualities. There's folklore going back to the first century A.D., when spider webs were used as dressings for wounds. Twenty-first century experts are looking at silk for many of the same reasons.

"So there are a lot of applications," continues Lewis. "People are interested in them for things like artificial ligaments and artificial tendons, bulletproof vests and even car airbags--something that would allow you to be contained, but not blown back in your seat."

But, whether it's for super-strong sutures for surgery or an air bag, how do you come up with enough raw material? Spider farms have been tried, but arachnids tend to kill each other.

"The problem is that the spiders are territorial, and so no matter what you do, there are only a certain number of spiders you can put in a certain space," says Lewis.

Key ideas:

• There is a lot of interest in spider silk fibers because they're stronger than almost any other manmade fiber
• Spiders are not farm-friendly animals which makes their silk difficult to mass produce

Sources
O'Brien, Miles, and Marsha Walton. "Research Areas." Nsf.gov. N.p., 03 May 2010. Web. 05 Oct. 2014. <http://www.nsf.gov/news/special_reports/science_nation/spidersilk.jsp>. 
Fecht, Sarah. "6 Spider-Silk Superpowers." Popular Mechanics. N.p., n.d. Web. 8 Oct. 2014. <ww.popularmechanics.com/science/health/med-tech/6-spider-silk-superpowers#slide-1>.

Paraphrasing

Spider silk has generated lots of interest because they are stronger than almost any other manmade fiber. It is stronger than steel and more elastic than a rubber band. Because of that, spider silk has sparked dozens of ideas of new and innovative ways of how to use it but nobody has had a sufficient amount of spider silk material to conduct full scale tests of these would-be products. Some possible products include lighter, stronger bulletproof clothing, artificial skin, better bandages, gentler airbags, elastic ligaments, and surgical stitches. Figure 1 below shows a spider weaving its web out of spider silk in the wild.

Figure 1: A spider making a web out of spider silk
http://www.popularmechanics.com/cm/popularmechanics/images/mG/Spider-Silk-01-0112-lgn.jpg

People have tried making spider farms to harvest spider silk but so far, nobody has been successful. This is due to the fact that spiders tend to be murderous and cannibalistic to each other. This makes their silk difficult to mass produce and harvesting it from the wild  is a very laborious and time consuming process.

So, how can we get the amazing powers of spider silk without having to harvest it from the wild? The answer to this question is goats - and not just any ordinary goats but transgenic goats.

The Specifics

General topic: GMO/transgenic organisms

More specific topic: Transgenic goats

Research question: How do you harvest spider silk from transgenic goats?

Problem: It is hard to harvest spider silk from spiders

Transgenic Animals - The Basics

Find the answer for following questions:

a.     In brief describe about this application (what is it all about?)

Transgenic animals are animals that have been genetically modified through purposely introducing foreign DNA material from another species. This foreign DNA is often times introduced through recombinant DNA technology and must be passed along the germ line so that all of the cells in the body will have the new genetic material.

b.     What are some benefits of this application?

Some benefits of transgenic animals include contribution to human welfare in three main areas: agriculture, medicine, industry and it a more viable way to create desirable traits in animals compared to selective breeding.

c.     What are some future improvements which still need to be done to make this application perfect (limitations)?

Some future improvement or limitations of transgenic animals is that the animals typically have a low survival rate, the procedure is expensive, it is a lengthy process, it can lead to abnormalities in the animal's body and transgenic animals could escape into the natural environment, which is very dangerous for the ecosystem. As you can see, the application of recombinant DNA technology to create transgenic animals still have a lot of future improvements to do in order to make it anywhere near perfect.

d.     What issues (social, economic, ethical, political or cultural) are currently related to the use of this application worldwide?

      There are some ethical issues of transgenic animals especially concerning those transgenic animals which are purposely flawed to research about diseases on because this will impact the animal's welfare and it also violates the animal's rights. There are also some religious issues but although most are against transgenic animals, there are actually some people in favor of it. In addition to that, transgenic animals also bend the food laws of some religions because even though an animal appears to be one species, it may have a part of a forbidden species in it.

Transgenic pigs

Sources:

• BBC News. BBC, n.d. Web. 05 Oct. 2014. <http://www.bbc.co.uk/ethics/animals/using/biotechnology_1.shtml>.
• Margawati, Endang T. "Transgenic Animals: Their Benefits To Human Welfare." Actionbioscience. N.p., n.d. Web. 06 Oct. 2014. <http://www.actionbioscience.org/biotechnology/margawati.html>.
• Ormandy, Elisabeth H., Julie Dale, and Gilly Griffin. "Current Context of Genetically Engineered Animals." National Center for Biotechnology Information. U.S. National Library of Medicine, 30 Oct. 2005. Web. 05 Oct. 2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078015/>.
• "TRANSGENESIS." Transgenic Organisms. N.p., n.d. Web. 06 Oct. 2014. <http://www2.wmin.ac.uk/~redwayk/lectures/transgenic.htm>.