Overview of Nexia's Process

The purpose of this section of our web page is to provide you an overview of the processes which are involved in producing a transgenic product such as Protexia™. This section of the website is for educational purposes only and, in the simplification of certain complex concepts, may not be accurate scientifically. For more information, please consult a genetics or molecular biology textbook.

Producing the Genetic Construct

Ultimately, Nexia wants to have the protein produced in the milk of its goats. To do this, Protexia™'s gene, must be placed in the right biological context to function properly. A gene has two main elements: the regulatory elements, which can be found at the beginning and end of the gene, and the gene itself. A gene can be thought of as being similar to a train, all trains have engines that steer the train in the right direction, and all trains have cabooses to signal the end of the train. Genes are much the same; they have to have the right "engine" and "caboose" to work properly. Getting the context right for the gene of interest is the field of molecular biology. Molecular biologists are experts in "cutting" genes and "fusing" them together in different environments and subsequently testing them to see if the new genetic construct works.

Gene splicing is one of Nexia's first steps in generating transgenic goats. The development of a genetic construct containing the gene of interest (e.g. human butyrlychanesterase genes) and its necessary surrounding sequences is critical for developing a transgenic goat. The gene must be turned on and off at the right time and must be produced, or "expressed", only in the mammary gland cells of the goat during lactation. Thus, Nexia uses a "milk-specific" promoter which directs the expression of Nexia's protein only in the mammary gland during lactation. Nexia has world renowned scientists specialized in molecular biology working to produce the best genetic constructs for our projects. Much of this process has been perfected for our transgenic goat system and is an essential part of our technology platform. In December, 2002, two of Nexia's scientists, Dr. Costas Karatzas and Dr. Anthoula Lazaris, were recognized as Team Leaders in Chemicals & Materials by Scientific American's Top 50, as leaders in innovation.

Preliminary Screening

After we have a genetic construct, we need to make sure that it is able to produce a functional protein. This can be done artificially, in vitro, using cell culture, or it can be done directly in animals, in vivo. Nexia prefers to do a preliminary screening process in vitro because it saves time and reduces the number of animals needed in our transgenic program. To perform the preliminary screening, we place the genetic construct into our patented MAC-T (Mammary Alveolar Cells with Large-T antigen) cell line. Nexia's proprietary MAC-T cell line is the only patented, non-human mammary cell line model world-wide. Nexia also ensures that the genetic construct is made properly by sequencing the DNA in the construct to ensure that the sequence is intact.

The screening in MAC-T step saves Nexia much time as it could take as much as three to five times longer to perform this step using goats alone. Moreover, using this method, Nexia reduces the number of transgenic goats that it must use.

In a parallel approach to using MAC-T cell line as a primary screening tool, we can test the genetic construct in a mouse model to verify that our proteins are being expressed properly in milk. Mice only generate a very small amount of milk and therefore are not appropriate for production purpose.

Transferring the Genes

Once a genetic construct is deemed to be expressed adequately in our screening systems, the construct can be transferred to the goat using one of two different methods. The first method uses microinjection of the genetic construct into a fertilized egg. This traditional method of pronuclear microinjection involves literally injecting the genetic construct into the pronucles of a one-celled fertilized egg with an extra-fine needle. The genetic construct will merge itself with the fertilized egg's native DNA and once this is achieved, the egg is transferred to a recipient goat and the pregnancy follows its normal course. The second method uses nuclear transfer technology (see Biology Fundamentals - Transgenics). In this method, the nucleus of a cell containing the genetic construct of interest is fused to a recipient fertilized egg whose native DNA has been previously removed and the resulting egg is transferred to a recipient goat. Once the transfer has occurred, the pregnancy of the recipient goat will progress as usual. Nexia's Assisted Reproduction Technology (ART) team is key in the process of generating and propagating the transgenic founder animals. This team has developed advanced reproductive techniques which are utilized for: oocyte recovery, zygote recovery, embryo transfer, semen production, and artificial insemination. Nexia's ART team has pioneered the proprietary process of using laparoscopic ovum pick-up (LOPU) for the generation of our transgenic goats using microinjection and nuclear transfer. Both microinjection and nuclear transfer techniques require a tremendous amount of technical expertise and much of this process has been perfected at Nexia.

BELE^® Goat System

Nexia uses a special type of goat for the generation of transgenic founders. These goats, Breed Early Lactate Early (BELE^®), provide Nexia with a shorter generation time due to a quicker time to reproductive maturity (see Biology Fundamentals - Goat Biology). BELE^® goats are able to produce commercial quantities of milk containing the protein of interest. The reduced time for production of meaningful quantities of protein can, in turn, speed the commercialization process.

After a transgenic line has been established, Nexia usually will breed the transgenic founder animal with a standard breed of goat, such as Saanen and Alpine. Nexia chooses to do this for two reasons. First, crossing into another breed of goat increases the genetic diversity, and second, standard goats produce more milk per year than BELE^® goats (see Biology Fundamentals - Goat Biology).

Milking and Purification

Milk is collected from our goats in a modern milking parlour. Once collection has occurred, the milk is treated using specialized processes to purify the protein from the milk. This involves skimming the milk and subsequently precipitating (aggregating) the protein out of the milk.