Working with Lentivirus for Generation of Transgenic Bovine Embryos

Abstract

Pharmaceutical industry started with the production of proteins for therapeutic purposes using the extraction from producing tissues, such as insulin derived from a pig's pancreas and growth hormone from the pituitary gland. The proteins from these tissues often show low quality and increase in the risk of transmitting diseases such as HIV or those caused by prions. Recombinant DNA technology has allowed the heterologous proteins production using the bacterial system [1] that results in increased protein availability, therefore, improving quality and reducing the risk of disease transmission. Bacteria have been widely used as bioreactors because they are easily cultured. However, these microorganisms have limited ability to perform post-translational modifications of proteins necessary for the formation of biologically active molecules [2].Mammalian cells achieve complex post-translational modifications, but the use of this system has a high cost for the large-scale protein production. Transgenic animals produce recombinant proteins with lower costs than mammalian cells. However, the establishment of production is relatively slow and still presents the same regulatory hurdles [3]. Mammary gland is considered the principal tissue for the expression of recombinant proteins and great efforts have been employed to generate transgenic animals for this purpose. Currently, two biopharmaceuticals derived from the milk of transgenic animals are commercially available: anti-α-thrombin (ATryn) produced by GTC Biotherapeutics UK Limited and C1 esterase inhibitor (Ruconest) produced by Pharming Group NV.Retroviruses have been used in different techniques of transgenic animal generation, such as nuclear transfer, embryonic stem cells, gene transfer mediated sperm and in the transduction of oocytes or zygotes. The size of the exogenous DNA to be inserted into the lentiviral vectors is the principal limitation of the technique, due to its packaging into a viral particle. Lentiviral transgenesis is based on the transfer of exogenous genes to embryos before implantation in the uterus. For efficient infection of mammalian zygote, the lentivirus must overcome the zona pellucidae, the extracellular matrix that surrounds the oocyte and embryo, forming a physical barrier against viral infection [4]. Two main methods to infect zygotes were reported, one consisting of subzonal injection of lentivirus in the perivitelline space [5][6][7][8], and lentivirus infection of denuded zygotes with zona pellucidae enzymatically digested [5,9].The success of these methodologies was obtained using the commercial vector FUGW. Therefore, we have adopted this vector to produce the lentiviruses and use them as a control to the standardization of zygote transduction. We have constructed other lentiviral vectors by directing the recombinant protein expression in the milk [10] and used the lentivirus to promote zygote transduction.The lentivirus was produced in 293-FT cells, and then concentrated concentrated about 100-fold using PEG, before the transducti...

Human proinsulin production in the milk of transgenic cattle

“…The size of genomic material is limited for the correct packaging of the lentivirus; therefore, large constructs as that used by Qian et al 10 are not possible using lentiviral vectors, and a large amount of genomic material can interfere with integration into genomic cells. 29 The lentiviral construct was constituted by a partial bovine β-casein promoter followed by the human insulin gene from the start codon until the poly-A signal, containing exons and introns. Mass spectrometry analysis verified a low relative abundance of recombinant protein in transgenic milk compared to milk proteins.…”