Transgenic rabbits for the production of biologically-active recombinant proteins in the milk

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Abstract

The use of live bioreactors for the expression of human genes in the mammary gland of transgenic animals is one of the most cost-effective ways for the production of valuable recombinant therapeutic proteins. Among the transgenic species used so far, rabbits are good candidates for the expression of tens to hundreds of grams of complex proteins in the milk during lactation. The lactating mammary gland of rabbits has proven to be effective in the processing of complex proteins. In this work, the potential use of rabbits as bioreactors is discussed based on our results and the published data.

Introduction

Since the first transgenic farm animals were generated in 1985 [1], relatively little time has elapsed; however, important achievements have been made in the transgenic technology. Among those are: the creation of knock out mice [2]; pigs for the production of organs intended for xenotransplantation to humans [3]; farm animal species secreting complex drugs in their milk [4]; and more recently the birth of transgenic cloned ruminants from differentiated adult or fetal cells through nuclear transfer.

Among the areas in which the transgenic technology is expected to exert a powerful influence, the expression of recombinant protein genes in the milk of transgenic livestock is undoubtedly one the most developed at present. In 1987 Gordon and coworkers showed for the first time that transgenic mice could appropriately process a complex human protein gene like the tissue plasminogen activator gene [5]. At present, hundreds of lines of transgenic mice expressing recombinant proteins in their milk have been produced. The mice have served only as a predictive model for the generation of transgenic farm animals, however the choice criteria for selecting the most suitable species for gene farming is usually based on the quantity of protein needed per year. A simplified rule is: the production of a protein in tons should be carried out by cows, in hundreds of kg by sheep or goats, and in kilograms per year by rabbits. Transgenic rabbits fit well as an intermediate animal for the production of limited amount of proteins [6]. Rabbits have short reproductive interval, can be reared under specific pathogen-free conditions and their milk is relatively well characterized [7]. A transgenic lactating doe can yield up to 10 kg of milk per year under semi-automatic hygienic milking condition [8]. All these features make rabbits attractive for the mammary-gland specific expression of recombinant proteins [9], [10].

Section snippets

Factors affecting the efficiency of generation of transgenic rabbits

The success of gene transfer in rabbits can be expressed as: (1) the survival rate (animals born/injected embryos transferred); (2) the transgene integration frequency (transgenics/no. of offspring); and (3) the overall efficiency (transgenics/injected embryos). The values of these figures vary between gene constructs and the average success rates obtained in rabbits are 10–15% survival rate, 8–12% integration frequency, and 1–2% efficiency [6]. It is important to point out that gene constructs

The need of cellular models

One of the main hurdles of current mammary gland transgenesis is the lack of appropriate cellular models for testing transgene constructs before spending time and effort to generate transgenic mice or farm animals. Mice have acted as laboratory models for testing gene constructs, however, as shown by multiple reports [12], [27] the predictive potential of the transgenic mice is very limited. Therefore, cell models, although are not a mirror of physiological processes of whole animals, are of

Production of proteins of high value by transgenic rabbits

The possibility of establishing transgenic mice and farm animals has opened new ways for the production of pharmaceutical proteins. The advantages of this method are that the production of proteins in vivo is more accurate and efficient than in vitro. The arising costs are 5–10 times lower than in tissue culture. The mammary gland is the most interesting organ for the production of recombinant proteins (for reviews see refs. [31], [32], [33], [34]. It has an enormous physiological potential for

Expression in the mammary gland

In rabbits, caseins are the major protein constituents of milk and their concentration in rabbit’s milk is above 60 mg/ml [35]. In the whey fraction, the WAP is abundantly represented and might account for at least 15% of the total milk mRNA. WAP is expressed at 1 5 mg/ml in the milk of the rabbits [35]. The promoters of all these major milk protein genes have been cloned, studied, and used for the expression of transgenes in the mammary gland of mice and rabbits (Table 1; for a review see

WAP promoter

Rabbit and mouse WAP-controlling sequences have been extensively used for the generation of transgenic mice and rabbits. Despite giving high expression levels of transgenes in the mammary gland in most of the cases studied so far, the WAP promoter also have elicited ectopic expression in the transgenic animals. In one study, murine WAP was expressed ectopically in transgenic sheep [38]. In general WAP-derived transgenes have shown erratic behavior that includes precocious transgene expression,

Other whey gene promoters

Regulatory elements from other whey genes have been employed in transgenic rabbits. In line with the efforts to express hEPO in the milk of transgenic animals, bovine β-lactoglobulin-hEPO fusion protein was expressed in rabbit’s milk under the control of bovine β-lactoglobulin promoter fragment. Expression levels were higher than in all the above discussed reports, but were still low (0.5 mg/ml). The fusion construct included a cleavage site for bacterial IgA protease. Although expression was

αS1-casein promoter

αS1-Casein is present at high concentration in the milk of most mammals, including rabbits [49]. For this reason its regulatory sequences have been extensively used to target the expression of recombinant genes in the mammary gland of transgenic animals. We generated transgenic mice and one transgenic rabbit founder from an expression cassette composed of a 1.6 kb of the bovine αS1-casein promoter fragment fused to cDNA encoding the htPA [50]. The expression levels were in the order of 50 mg/l

Acknowledgements

We thank Dr Bernd Groner for laboratory space and support, Ch. Beisenhaus for purified Stat5, Olivier Sandra and Alexis Schubert for encouraging support, Yangtsé Portelles for isolating FY-2 cells, Dagmara Pichardo for animal care, Jesús Seoane and Nilda Reyes for the artwork. Part of this work was supported by a Deutscher Akademischer Austauschdienst (DAAD) fellowship to FOC.

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