Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin

Abstract

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79×10−2 percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.

Allergenicity Assessment of a Genetically Modified Protein-Recombinant Human Lactoferrin

“…In previous studies of the rhLF transgenic cows the composition of milk and milk powder from transgenic cows were compared to that from non-transgenic cows. The results did not demonstrate any significant differences with the exception of the presence of high levels of rhLF in the transgenic milk [32,33]. Biochemical characterization of rhLF and hLF demonstrated slight differences in molecular weight, with rhLF being slighly lower than hLF.…”

“…Transformation was accomplished through microinjection of a bacterial artificial chromosome (BAC) containing a copy of genomic clone (~150 kb) of hLF into bovine fibroblasts, followed by somatic cell cloning and transfer to the uterus of recipient cows with a small number of transgenic calves being produced [32]. In previous studies of the rhLF transgenic cows the composition of milk and milk powder from transgenic cows were compared to that from non-transgenic cows.…”

Transgenic bovine as bioreactors: Challenges and perspectives

“…Transgenic cattle have been generated using this vector. 12 Plasmid vectors are extrachromosomal, self-replicating DNA molecules. Many of the plasmid vectors are only 3 to 5 kb in length.…”

“…The methods investigated include recloning, autologous SCNT, impeding Xist expression from the active X chromosome, treatment with trichostatin A after SCNT and histone deacetylase inhibitors, among others. SCNT has been successfully used for the generation of transgenic cattle for recombinant protein expression in milk aimed at nutritional 12,14 and therapeutic 50,51 purposes. Effective SCNT can be applied to the production of transgenic animals that could contribute toward the development of sustainable and diversified animal production systems.…”

Expression and characterization of a lysine rich protein in cow milk

“…It may be useful to remove allergenic proteins and to reduce lactose concentration (Jost et al, 1999;Vilotte et al, 2002). Some alleles of milk protein genes may be added to improve curd quality or to increase total milk nutritional value (Brophy et al, 2003;Yang et al, 2008). The milk may also be a carrier to provide consumers with anti-pathogen molecules (Soler et al, 2005) or with high quality lipids (Kao et al, 2006).…”