8.1: Applications of Animal Biotechnology

Biotech Focus

What comes first, the chicken or the egg?

Well, thanks to biotechnology and precision fermentation, turns out its neither one of them. Precision fermentation is an emerging food technology that uses microbes, like yeast, to produce specific functional food ingredients, some of which traditionally require farm animals. One target of this technology is the egg. The "white" of a chicken egg is 88% water, 10% protein (mainly ovalbumin), and trace minerals and carbohydrates. At EVERY Company, scientists use genetically-modified yeast cells to produce the ovalbumin found in chicken egg whites. The yeast are genetically-modified with the gene for chicken ovalbumin and grown in large fermenting tanks. Today, the company uses this technology to produce egg white proteins without the hen. What results is called the EVERY Egg^TM - the world's first "henless" egg. So similar is the EVERY Egg^TM to the real thing, EVERY Egg^TM debuted in 2023 at Eleven Madison Park in New York City, a three-star Michelin restaurant considered among the world’s best of the best.

To learn more about precision fermentation, check out the article The Eggs of the Future will be from Precision Fermentation.

Introduction

Animal biotechnology is a branch of biotechnology in which recombinant DNA techniques are used to genetically engineer (i.e. modify the genome of) animals in order to improve their suitability for use in the livestock industry, medicine, or pharmaceuticals. The last decade has seen significant advances in animal biotechnology, with researcher making progress in sequencing animal genomes, gene expression, and the proteomic analysis of animal cells. Through their work, animal biotechnology has lead to increased food production from healthier, faster-growing livestock, increased disease resistance as engineered animals are less vulnerable to infections, improved breeding using assisted reproductive technologies, and medical breakthroughs through transgenic animals producing human medicines.

Learning Objectives

Animal biotechnology genetically engineer animals to improve their use in the livestock industry, medicine, and pharmaceuticals. At the end of this section, you will be able to

• Describe how genetic engineering (gene addition, gene silencing) is used to improve the health of animals and their productivity
• Describe how biopharming can be used to produce compounds beneficial to humans
• Describe how liposomes and nanoparticles are an alternative to viral vectors in gene delivery
• Describe how CRISPR works in gene editing
• Describe the types of cell cloning

Techniques & Applications of Animal Biotechnology

Animal biotechnology uses specific biological techniques and tools. Some of these techniques include genetic engineering, gene editing, and cell cloning. Their applications in animal biotechnology can be wide and varied.

Genetic Engineering

Genetic engineering uses recombinant DNA techniques to introduce, remove, or modify genes within an organism's genome. Several terms are used to describe genetically engineered animals, including genetically-modified, genetically-altered, genetically-manipulated, and transgenic. For an introduction to genetic engineering and the tools used for this field, see Chapter 4: Genetic Engineering & Recombinant DNA Technology.

The two principle methods in genetic engineering are gene introduction/addition and gene silencing. The goal of gene addition is to introduce a functional copy of a desired gene into the genome of an organism. In humans, this gene is usually added in order to replace a defective or missing one. However in animals, gene addition is most often used to introduce a new trait to an animal or improve upon an existing one. The new or modified trait(s) can improve the health of a species, enhance its nutritional profile or its productivity, or enable the use of animal products for use by humans. Examples include the addition of genes that make pigs resistant to Porcine Reproductive and Respiratory Syndrome (PRRS), or genes to cows to protect them from Bovine Spongiform Encephalopathy (BSE) (i.e., mad cow disease). Increasing disease resistance using gene addition in endangered animals is seen as a promising way of improving the health and survival of these animals. Gene addition has also been suggested as a means of re-introducing extinct animals, such as the woolly mammoth, by inserting mammoth genes into the elephant genome. Commercially, the addition of the Pacific Chinook salmon growth hormone gene, in combination with a promoter from ocean pout (an eel-like fish) into Atlantic salmon has resulted in the production of AquAdvantage® salmon, a genetically engineered salmon with a two-fold increase in growth rate when compared to their unmodified counterparts. Regulatory genes have also been added to the genomes of cattle, pigs, and sheep in order to increase their muscle mass, thus decreasing the time it take for these animals to come to market. Outside of its commercial applications, gene addition has be used to insert genes for the production of therapeutic compounds by a wide variety of animals for their use in medicine. For more information, go to Chapter 8.2 Genetic Engineering in Animals.

Viral vectors, such as adeno-associated virus (AAV) or lentivirus, can be modified to introduce genes into animal and human cells without causing disease. Viral vectors have the advantage of being able to deliver the functional gene to a significant number of cells. However, the potential for a deleterious immune reaction can be high with the use of viral vectors. In humans, modified viral vectors are currently being using in the treatment of sickle cell anemia, blindness and spinal muscular atrophy (SMA). While the results are promising, the cost of viral-based gene therapy is very high. As an alternative, genes can be introduced using synthetic delivery methods like liposomes and nanoparticles. Liposomes and nanoparticles can be used to deliver genes to cells owing to their ability to encapsulate DNA or RNA and protect them from degradation in the bloodstream. The attachment of specific ligands to nanoparticles so that the delivery of genes can be targeted to a specific cell type has shown promise in enhancing treatment efficacy and minimizing side-effects. Encouraging results have been observed using targeted nanoparticles for the delivery of apoptotic genes to lung cancer cells. However, like viral vectors, the immune system can decrease the effectiveness of nanoparticle delivery. In addition, the percentage of cells receiving these nanoparticles is significantly lower versus viral vectors.

In gene silencing, a defective gene is "turned off" inside of a cell. The most common way of doing this is through the use of RNA interference (RNAi) technology. RNAi mechanisms within a cell target specific mRNA molecules for degradation or inhibit their expression into proteins. RNAi molecules, including small interfering RNA (siRNA) and microRNA (miRNA) can be delivered and introduced into cells using nanoparticles, antibodies or modified viruses. RNAi molecules can also be attached to cell surface molecules for internalization. The first commercial RNAi-based therapeutic, approved by the FDA in 2018 is sold as ONPATTRO® for the treatment of hereditary amyloidogenic transthyretin (hATTR) amyloidosis. Today, the gene therapy using RNAi is being explored in the treatment of Huntington’s disease and certain cancers. For more about gene silencing and RNAi, read Chapter 5.5: Functional Genomics.

Gene Editing using CRISPR

Gene editing allows for the precise and direct modification of a genome by adding, removing, or altering DNA at a specific location. Like genetic engineering, gene editing modifies an organism's genome to change a specific trait or characteristic. These modifications can include gene addition or gene silencing. However, gene editing typically does not introduce foreign DNA into a host's genome but uses DNA from the same species. One powerful method for gene editing is the technique known as CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR is derived from a bacterial defense mechanism that protects bacteria from the integration of viral DNA (from bacteriophage) into the bacterial genome. For this mechanism, bacteria evolved a location in the genome called a CRISPR locus. The CRISPR locus is made of short regions of repetitive bacterial DNA known as direct repeats (Figure \(\PageIndex{1}\)). In between these repeats, the bacteria inserts short sequences of bacteriophage DNA (approximately 20-50 nucleotides each) known as "spacers". These spacers serve as a "memory" of previous bacteriophage infections and act as future defense against another invasion by the same bacteriophage. Transcription of the CRISPR locus followed by RNA processing produces several pieces of CRISPR RNA or crRNA. Each crRNA will contain one of the bacteriophage spacers. The bacteria also produces an additional piece of RNA called tracrRNA, which becomes associated with a type of bacterial nuclease called a CRIPSR-associated nuclease (Cas). Bacteria express several Cas isoforms. However, the most commonly studied one is Cas9. Upon infection of the bacteria with a bacteriophage, the tracrRNA matches up the crRNA with its corresponding piece of bacteriophage DNA. When this match occurs the Cas9 enzyme cuts the bacteriophage DNA, rendering it inactive.

Figure \(\PageIndex{1}\): The CRISPR system of bacterial immunity. Adaptive immunity against bacteriophage infection is achieved in bacteria through the CRISPR system. The CRIPSR locus is comprised of several palindromic repeats of DNA (DNA repeats, black squares) found in between regions of integrated bacteriophage DNA known as spacers (red, green and yellow triangles). The CRISPR locus also contains genes for several isoforms of the Cas enzyme (Cas gene, blue arrows), the gene for tracrRNA (red arrow), and a leader sequence (not shown) that plays an essential role in transcription of the spacers and DNA repeats. CRISPR locus gene expression produces several Cas isoforms, of which Cas9 plays an essential role in immunity. Transcription from the locus also produces tracrRNA (red line), and a piece of "pre-crRNA" comprised of the spacers and DNA repeats. CRISPR assembly results in the hybridization of the tracrRNA with its complementary sequence found in the pre-cRNA and the binding of the Cas9 to the pre-cRNA as well. Processing by an RNAse, cuts the pre-cRNA into several pieces of crRNA (i.e., a spacer and a DNA repeat), each associated with a piece of tracrRNA, and a Cas9 protein. Upon infection by a bacteriophage, the spacer in the crRNA binds to its complementary region in the bacteriophage DNA. The Cas9 enzyme cuts the bacteriophage DNA at that region, rendering it inactive. (CRISPR by Patricia Zuk, CC BY 4.0)

Since its discovery, the CRISPR system has been modified to edit the genome of mammalian cells in the laboratory. For this, researchers combined the tracrRNA and crRNA into a single guide RNA (sgRNA) that retains the functions of both its crRNA and tracrRNA components. This sgRNA can be designed in the lab to match up with a specific sequence within the mammalian cell's genome where DNA changes are desired. The Cas enzyme is provided to the cell via a DNA plasmid for gene expression or directly as purified protein. The sgRNA directs the Cas9 enzyme to the targeted DNA sequence where it will unwind a short stretch of host DNA. The RNA in the crRNA component can then bind to its complementary DNA sequence. The Cas9 enzyme cuts both strands of the host's DNA helix at that site. Sensing damage to its genome, the cell will repair the cut. However, because mammalian DNA repair is error-prone, the cell will often introduce mutations to the repaired site. If this area of the genome is a gene, the gene with the error will likely be inactivated. As such, CRISPR is often used to "knock-out" gene function.

CRISPR can also be used to excise a mutated section of a gene and replace it with the correct sequence. This can be done if an exogenous piece of DNA is provided to the cell during the repair process. If an entire gene sequence is provided to the cell during repair, the cell can insert this gene. In more advanced applications, Cas9 can be fused to other enzymes for delivery to a DNA sequence. Once there, these enzymes can modify the DNA sequence directly, producing mutations like stop codons to inhibit gene expression or changing the sequence of a mutated gene back to the correct sequence. While a powerful piece of technology, the efficiency of CRISPR gene editing is not 100% so correctly edited cells must be identified. Additionally, scientists are continuing to study the incidence of non-intended edits to the genome when using CRISPR-based systems.

Concept in Action

Animation: CRISPR - Gene editing and beyond

Cloning and Somatic Cell Nuclear Transfer (SCNT)

Somatic Cell Nuclear Transfer (SCNT), otherwise known as "cell cloning", transfers a diploid (2n) nucleus from a donor cell into a recipient egg in which the nucleus has been removed (i.e., enucleated)(Figure \(\PageIndex{2}\)). The genetically-modified egg is programmed by the egg's cytoplasm to begin its development into an embryo that is genetically identical to the donor cell. As such, this early stage embryo is often referred to as a clone. In reproductive cloning, the clone is implanted, through embryonic transfer, into a surrogate mother for continued development and delivery. Reproductive cloning has been employed to produce genetically identical copies of animals that have exceptional traits, such as enhanced milk production. Despite a high failure rate, numerous animals have been cloned and delivered using this method, including Dolly the sheep in 1996. Reproductive cloning holds promise for the conservation of extremely endangered animals and researchers have also proposed that Reproductive cloning could be used to "resurrect" extinct animals using frozen cells and suitable surrogate mothers (e.g. the woolly mammoth). In therapeutic cloning, the clone is cultured and allowed to develop to the blastomere stage (or blastocyst stage for human embryos). Embryonic stem cell (ES) cells are retrieved from this stage. The ES cells are used in regenerative medicine for the development of new tissues and organs.

Figure \(\PageIndex{2}\): Somatic Cell Nuclear Transfer (SCNT). The diploid nucleus from a somatic cell with the desired genes is removed and inserted into a recipient egg cell in which the haploid nucleus has been removed. The genetically modified egg cell is then allowed to undergo development to form an early stage embryo that is known as a clone. In reproductive cloning, the clone is implanted into a surrogate mother for continued development and delivery. In therapeutic cloning, the clone is placed into cell culture in order to produce stem cells that can be used in downstream applications, such as regenerative medicine. (SCNT by Patricia Zuk, CC BY 4.0; adapted from Somatic Cell Nuclear Transfer by Wikibob, CC BY-SA 3.0)

Concept in Action

Video: Microinjection of Nuclei for SCNT

Both reproductive and therapeutic cloning have raised concerns about cloning humans and the destruction of human embryos for the isolation of human ES cells. Today, most countries have stringent legal and ethical rules surrounding SCNT and cloning.

Key Concepts

The techniques used in animal biotechnology are wide and varied and include advanced techniques like CRISPR for gene editing and SCNT for cell cloning.

Some important concepts to remember are:

• genetic engineering and gene editing use recombinant DNA techniques to introduce, remove, or modify genes within an organism's genome
• gene editing allows for the precise and direct modification of a genome by adding, removing, or altering DNA at a specific location
• foreign DNA is usually added through genetic engineering; non-foreign DNA is added through gene editing
• genetically engineered organisms are referred to a transgenic organisms
• gene addition introduces a functional copy of a desired gene into the genome of an organism
• gene silencing inhibits the expression of a defective gene inside a cell

Glossary

 

Animal biotechnology – the application of genetic engineering, cloning, and molecular biology to modify animals for agriculture, medicine, and industry

Biopharming  – the use of genetically-engineered plants or animals to produce valuable medical or therapeutic compounds such as drugs, vaccines and antibodies; also called pharmaceutical farming or "pharming"

Cloning – the process of creating a genetically identical copy of an organism, often using somatic cell nuclear transfer (SCNT)

Cell cloning - also known as somatic cell nuclear transfer (SCNT)

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) - a gene-editing tool that uses a guide RNA and a Cas9 enzyme to find and cut specific DNA sequences

Disease-resistant animals – genetically modified or selectively bred animals that are less susceptible to infections

Embryo Transfer (ET) – a reproductive technology where fertilized embryos are transferred into a surrogate mother to improve breeding efficiency

Gene editing – the modification of an cell’s DNA to introduce beneficial traits; uses technologies like CRISPR

Genetic engineering - the direct modification of an organism’s DNA using biotechnology; often done to introduce new traits, to improve resistance to disease, or to produce beneficial products 

Genetically Modified Organism (GMO) - any organism whose DNA has been altered using genetic engineering techniques

Gene silencing - the inhibition of a gene's expression through natural or artificial means; can be achieved in the laboratory using siRNA or miRNA techniques

Genome - the entirety of the genetic information in a cell

Enucleated - a cell in which the nucleus has been removed; used in cell cloning

Liposome - a spherical vesicle made of phospholipids that mimics the structure of a cell membrane; used in transfection of mammalian cells

Recombinant DNA (rDNA) technology – techniques that combine DNA from different sources, used to create genetically modified animals

Reproductive cloning – cloning that uses SCNT technology to create identical animals for research, agriculture, or conservation

RNA interference - a lab technique used to target specific mRNA molecules for degredation; uses wither siRNA or miRNA molecules to affect mRNA stability and expression

Somatic cell nuclear transfer (SCNT) - a process in which a diploid (2n) nucleus from a donor cell is transferred into an enucleated recipient egg

Transgenic animal – an animal that carries genes from another species, typically used for research, medicine, or agriculture

Therapeutic cloning - cloning that uses SCNT technology for the isolation of embryonic stem cells

Xenotransplantation – the process of transplanting cells, tissues, or organs from one species to another, typically from animals to humans