Scientists producing transgenic1 cows use a range of techniques including DNA2 cloning, restriction enzyme3 digests, ligation4, polymerase chain reaction5 (PCR), transfection, nuclear transfer and in vitro6 embryo7 production.
Scientists at AgResearch have successfully generated transgenic cows that produce extra proteins in their milk using the 7 steps described below.
Step 1. Designing the gene construct
The first step is to design a gene construct. The gene8 construct is a unit of DNA that includes:
- an antibiotic resistance gene – to select cells that have taken up the gene construct
- a tissue-specific promoter sequence – to signal the start of expression of the protein in cells of the appropriate tissue, for example, in mammary cells in lactating cows
- the desired gene – for example, bovine casein or human myelin basic protein9
- a stop sequence – to define the end of the information for making the protein.
Step 2. Sourcing the transgene
In the past, the gene would have been extracted from the source organism’s DNA. Now, however, if the desired gene sequence is known, it can simply be synthesised in a lab. There are companies that make genes10 to order within a couple of weeks.
Step 3. Making the gene construct
The gene is usually supplied in a vector. A vector11 is a small piece of DNA, often a plasmid, into which a foreign piece of DNA can be inserted. When the gene of interest is in a vector, it can be sent from one lab to another, it can be stored, it can be manipulated or it can be used to transform bacteria to produce more copies of the gene of interest.
Vectors12 have multiple restriction enzymes sites (also called multiple cloning sites) so the gene can be inserted into the vector and then cut out from the vector using restriction enzymes13. This article has more information on restriction enzymes.
After the gene is cut from the vector, it is then pasted into the multiple cloning site of the gene construct using a method known as ligation. This article has more information on DNA ligation.
Step 4. Transfecting bovine cells
The gene construct is incorporated into the genome of a bovine (cow) cell using a technique called transfection. During transfection, holes are made in the cell14 membrane that allow the DNA to enter. The holes can be made by applying an electrical pulse or by adding chemicals15 to the cells. Once inside the cell, the gene construct may enter the nucleus and incorporate into the cell’s genome16.
Step 5. Selecting for transgene positive cells
After transfection, an antibiotic17 is added to select the bovine cells that have incorporated the gene construct. Transgenic bovine cells will survive treatment with an antibiotic, because they contain an antibiotic resistance18 gene making them resistant to the antibiotic. Cells without the gene construct will have no resistance to the antibiotic and will die. In addition to antibiotic selection, polymerase chain reaction (PCR19) is used to check that the bovine cell contains the transgene.
Step 6. Making a transgenic embryo using nuclear transfer
Nuclear transfer is used to create a whole animal from a single transgenic bovine cell.
The transgenic bovine cell is fused with a bovine oocyte that has had its chromosomes20 removed (called an enucleated oocyte). An electrical pulse is applied to help fuse the cells. Once fused with the oocyte, the transgenic cell’s chromosomes are reprogrammed to direct development into an embryo. After 7 days, the transgenic embryo will have about 150 cells and can be transferred into a recipient cow for further development to term.
Step 7. Confirming the cow is transgenic
If the embryo develops to full term, after 9 months, the cow will give birth to a calf. To confirm that the calf is transgenic, scientists can check using:
- PCR to determine the presence or absence of the transgene21
- quantitative22 PCR (q-PCR) to determine the number of copies of the transgene
- fluorescent in situ hybridisation (FISH) to visualise where the transgene is in the chromosome and whether the transgene has integrated into more than one chromosome23.
When the calf is lactating (either after being induced to lactate or after having its own progeny), its milk is checked to determine if the transgenic protein is being expressed.
Useful links
This video describes two methods for developing transgenic mice to study human genetic24 diseases25 and is from the BioInteractive website.
Nuclear transfer animation is a video animation of the nuclear transfer process, from the BioInteractive website.
- transgenic: An organism (plant, animal, bacterium or virus) whose genome has been changed using genetic material from a different species.
- DNA: Deoxyribonucleic acid (DNA) is a molecule that contains the instructions needed for an organism to develop and function. These instructions are stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C) and thymine (T).
- restriction enzyme: An enzyme that causes a break, or cut, in both strands of DNA at a specific sequence, usually about 4-8 base pairs long. Also called a restriction endonuclease.
- ligation: The joining together of DNA fragments using ligases.
- polymerase chain reaction (PCR): A method that rapidly increases the number of copies of a target DNA sequence. Can be used for detecting small amounts of DNA material or generating multiple copies for use in further processes.
- in vitro: Occurring in a laboratory environment, for example, in a test tube. This is a Latin term meaning ‘in glass’.
- embryo: The product of a fertilised egg, from the zygote until the foetal stage. The undeveloped plant that forms when the ovule is fertilised.
- genes: A segment of a DNA molecule that carries the information needed to make a specific protein. Genes determine the traits (phenotype) of the individual.
- protein: Any of a large class of complex compounds that are essential for life. Proteins play a central role in biological processes and form the basis of living tissues. They have distinct and varied three-dimensional structures. Enzymes, antibodies and haemoglobin are examples of proteins.
- genes: A segment of a DNA molecule that carries the information needed to make a specific protein. Genes determine the traits (phenotype) of the individual.
- vector: Biology: a carrier, i.e. a mosquito is a vector for malaria. Biocontrol: a vector carries the control agent to the target organisms, i.e. blowflies (vector) spread calicivirus amongst rabbits. Genetic engineering: a tool used to carry a gene of interest. Vectors are small pieces of DNA, often a plasmid, and are used to carry foreign DNA into a cell. Physics: a quantity that has both magnitude and direction.
- vector: Biology: a carrier, i.e. a mosquito is a vector for malaria. Biocontrol: a vector carries the control agent to the target organisms, i.e. blowflies (vector) spread calicivirus amongst rabbits. Genetic engineering: a tool used to carry a gene of interest. Vectors are small pieces of DNA, often a plasmid, and are used to carry foreign DNA into a cell. Physics: a quantity that has both magnitude and direction.
- enzyme: A complex protein that acts as a catalyst (speeds up chemical reactions) in specific biochemical reactions. For example, saliva contains an enzyme called amylase that can break down starch into simple sugars.
- cell: 1. Building block of the body. A human is made of millions of cells, which are adapted for different functions and can reproduce themselves exactly. 2. A simple electrolytic device that enables chemical energy to be transformed into electrical energy.
- chemicals: Everything is made up of chemicals. All matter (anything made of atoms) can be called chemicals. They can be in any form – liquid, solid or gas. Chemicals can be a pure substance or a mixture.
- genome: The complete set of DNA in any individual organism.
- antibiotic: A substance produced by an organism that kills bacteria.
- resistance: 1. The opposition to the flow of electric current through a circuit. 2. The ability to withstand harmful or unfavourable conditions, such as an infectious disease.
- polymerase chain reaction (PCR): A method that rapidly increases the number of copies of a target DNA sequence. Can be used for detecting small amounts of DNA material or generating multiple copies for use in further processes.
- chromosome: A structure within the cell nucleus made of a single coiled piece of DNA that contains the genetic blueprint of an organism.
- transgene: A gene that has been integrated into the genome of a recipient organism.
- quantitative: Looking at numerical aspects of something; measuring and comparing something in units.
- chromosome: A structure within the cell nucleus made of a single coiled piece of DNA that contains the genetic blueprint of an organism.
- genetic: Of, relating to, or determined by genes.
- diseases: 1. An abnormal condition of an organism that impairs bodily functions. 2. In plants, an abnormal condition that interferes with vital physiological processes.