Online reference: Transgenic and targeted mutant animal database Example: "The Knockout of the Month" for June 2001: BACE (beta amyloid precursor protein cleaving enzyme) as a therapeutic target model for Alzheimers disease

Genetic engineering of animals

Regulation of gene expression

Remember our model for the typical eukaryotic gene:

You can get your feet wet, with:

Transient transfection of cells.

The way to find out if your DNA is being expressed is to introduce a "reporter gene," which encodes an easily assayed gene product, into your plasmid.

Stable transfection of cells

Eukaryotic cells tend to be sensitive to some antibiotics such as G418 (a derivative of Neomycin) and hygromycin, for which resistance genes are available. You can use your cloning techniques to place a resistance gene into the DNA from your highly transcribed region (deleting the native gene) and introduce that linearized DNA into cells, promoting homologous recombination. This is essentially the model we've discussed for T-DNA recombination into the plant genome.

The end result is that a drug resistance gene can be integrated into the genome, in place of a native gene:

You can design a PCR method to track the integration event, determining whether it is specific or nonspecific.

Fig 19.6 from book

Now if you grow your transfected cells in hygromycin, cells that are not expressing the Hyg resistance gene will be killed by the drug. Some of the cells that are hygromycin resistant may have integrated the gene by homologous recombination (as shown above) and others may have integrated it into another chromosomal locus.

Fig 19.7 from book

Positive/Negative Selection - an example, in the case of plasmid shuffle
There are many yeast vectors available that permit integration (the YIp type) or maintenance as an extrachromosomal element (the YEp type, which have origin of replication elements). Using these vectors, beautiful and elegant genetic experiments can be performed. An example is the "plasmid shuffle" experiment:

Let's suppose you've used your genetic engineering skills to make a mutated allele A for a gene of interest, and you want to introduce it into cells and have it be the only copy of the gene. If the gene is required for viability, how will you exchange the wild type for the A allele without killing the cell?

By this method, one plasmid has been exchanged for another, and consequently one allele of a gene has been exchanged for another.

An alternative use of the TK gene and ganciclovir is discussed in your book, figure 19.5: Positive-Negative Selection

Getting the DNA into the cell, and getting a founding breeder!

Microinjection methods

Fig 19.2 from book

What do you do with your potential mosaics? You put them on the stud farm!

Another approach, using nuclear transfer from a pluripotent cell line.

ES methods

Another approach - based on embryonic stem cells

Fig 19.4 from book

Retroviral methods

• Packaging helper cells.
• Pseudotyping methods for host and cell range control.
• What is the chance of creating replication-competent, recombinant retroviruses?

  

The goal is germ line modification.

Resource reading: Transgenic mice produced by retroviral transduction of male germ-line stem cells. Makoto Nagano*, Clayton J. Brinster, Kyle E. Orwig, Buom-Yong Ryu, Mary R. Avarbock, and Ralph L. Brinster. Proc. Natl. Acad. Sci. USA 98, Issue 23, 13090-13095, November 6, 2001

Resource reading: Transgenic Monkeys Produced by Retroviral Gene Transfer into Mature Oocytes. A.W.S. Chan, K.Y. Chong, C. Martinovich, C. Simerly, G. Schatten. Science 291 309-312. 12 January 2001

Resource reading: Generation of Transgenic Poultry by Transfection of Primordial Germ Cells. E.A. Wong, et al. in: Transgenic Animals in Agriculture.

Germinal crescent -> gonad

See also, blastoderm transfection, Figure 19.14

Pharming

How to make a transgenic cow - Fig 19.13

Animals as bioreactors (PPL Therapeutics Ltd.) "Like conventional E.coli based fermentation systems, transgenic production is capable of producing large quantities of material. The great advantage of PPL's transgenic approach is that it can do so with infinitely greater flexibility while eliminating many of the costs and limitations associated with traditional recombinant production. ... By using livestock as the bioreactors, production volumes can be scaled up or down easily and cheaply by adjusting the number of animals in the flock or herd, without the need for extra facilities. The capital costs associated with the maintenance of animals are also tiny in comparison to the cost of constructing fermentation facilities." source:http://www.ppl-therapeutics.com/Welcome/Products/Product8/product8.html

Application: Fibrinogen, as a fibrin sealant in wound repair - PPL Therapeutics Ltd.

Genzyme Transgenics Inc. "Genzyme Transgenics has demonstrated consistent expression levels for fusion molecules including immunoglobulin (Ig) fusions in the milk of transgenic animals in the 2 - 6 gram per liter range. Production of fusions require stable integration of two (or more) gene constructs, simultaneous expression of each protein and proper assembly into tetrameric protein complexes. Genzyme Transgenics has successfully produced more than seven Ig fusion proteins in the milk of transgenic animals. We have been successful in expressing Ig fusions containing antibody molecules linked to enzymes for in vivo targeting, single chain antibodies, hybrid molecules containing ligands attached to an immunoglobulin-like scaffold and fusions of receptors to antibody fragments (i.e. PRO542 for Progenics Pharmaceuticals). "source: http://www.transgenics.com/fusionplatform.html

Resource reading: Q&A at Genzyme Transgenics: "How does transgenic expression compare with other production methods? There are four other means of commercial protein production. E. coli production, which was the first commercialized, is very efficient, but limited to simple non-glycosylated proteins. Although the cost of production is low, the cost of processing and refolding the proteins is significant. Fungal systems, such as Pichia or filamentous fungi allow efficient production of some secreted proteins, but the glycosylation is usually high mannose which can effect the pharmokinetics of the protein. There is also the baculovirus production system, which can produce a wide range of proteins in small scale, but has yet to be scaled up to commercial levels. The standard method for producing complex glycosylated proteins, (i.e. Monoclonal Antibodies) is with cell tissue culture. The protein may be properly folded and modified, but the low yields per cost of production facility limit the number of proteins that can be developed. Recombinant protein concentrations in the milk of transgenic animals are substantially higher than levels attained in cell tissue cultures. Expression levels of 2 to 10 grams of recombinant protein per liter of milk are readily achievable in transgenic livestock. In comparison, highly optimized cell cultures can typically generate 0.2 to 1 gram per liter of culture medium." source: http://www.transgenics.com/science/questions.html

Got milk?

BioSante Inc. "BioSante Pharmaceuticals, Inc. has been recently issued a U.S. patent for the advanced method of selectively isolating biologically active therapeutic proteins from transgenic milk in a casein-free supernatant. Obtaining commercially viable yields of recombinant target protein from the milk of transgenic livestock remains a challenging task since milk is a complex, multiphasic (fat, casein, micelles, whey) colloidal suspension. We believe our patented process effectively addresses these well-known challenges and offers significant efficiency and economy compared to the currently employed methods."source: http://www.biosantepharma.com/products/milkpatent.html Resource reading: Milk Protein White Paper (5 pages, pdf file) Traditional approach: Fat removal (cream separation by centrifugation) Casein removal (pH 4.7 precipitation or chymosin treatment) leads to product loss Whey fraction (proteins, containing transgenic product) Alternatives PPL Therapeutics Ltd (makers of Dolly): Patented method for purification of alpha-1 anti-trypsin from sheep milk (up to 40 g/liter of milk, and one sheep can produce 500 liters per year) involving PEG precipitation of casein and PEG fractionation of whey fraction, followed by sequential chromatography methods. PPL does its work in New Zealand - a prion-free country. Figure 19.12 from book - beta-casein-CFTR fusion Genzyme Transgenics Inc: method involves dilution of raw milk, microfiltration, and chromatography. "Genzyme Transgenics' efforts are focused on the development of proteins for commercial use. Particularly, the company has developed substantial expertise and increasing focus on monoclonal antibodies for therapeutic use in chronic diseases such as rheumatoid arthritis, other autoimmune conditions and cancer." source: http://www.transgenics.com/about.html BioSante Inc: method involves fat removal and treatment with proprietary calcium phosphate "nanoparticles" or "CAP". This is said to reform the casein micelles "away from" the target protein, so that they may be removed by centrifugation. Net - 65-75% yield. CAP, which is considered GRAS by the FDA, may have other applications as a protein delivery vehicle in an insulin inhalant and as a vaccine adjuvant.

Report: PPL Therapeutic Ltd's recombinant AAT is delayed in phase III trials, as of March 18, 2002
And you can see what effect this has on stock price!

Nuclear Transfer

Figure 19.8 from book
PPL - nuclear transfer

Xenotransplantation

• Hyperacute rejection
• Delayed xenograft rejection
• Long term rejection

Resource reading: PPL - xenotransplantation