MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Monzani, Paulo Sérgio; Guemra, Samuel; Adona, Paulo Roberto; Ohashi, Otávio Mitio; Meirelles, Flávio Vieira; Wheeler, Matthew B.

doi:10.1080/10495398.2014.941468

Cited by 8 publications

(11 citation statements)

References 24 publications

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“…The transduction and selection of bovine fibroblasts and MAC-T cells were performed as previously described. 14,24 The fibroblast cultures were performed using DMEM-high glucose medium (Gibco # 11995065, Carlsbad, CA), 10% fetal bovine serum (Gibco # 26140079, Carlsbad, CA), 100 units mL −1 penicillin (Sigma-Aldrich # P3032, St. The primers were the forward 5′-GGCGTGAACCACGAGAAGTATAA-3′ and reverse 5′-CCCTCCACGATGCCAAAGT-3′.…”

Section: Bovine Mac-t Cells and Fibroblast Transductionmentioning

confidence: 99%

Human proinsulin production in the milk of transgenic cattle

Monzani,

Sangalli,

Sampaio

et al. 2024

Biotechnology Journal

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BackgroundThe worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk.Methods and ResultsPseudo‐lentivirus containing the bovine β‐casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin‐degrading enzyme that could degrade the recombinant protein.ConclusionThe methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

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Section: Bovine Mac-t Cells and Fibroblast Transductionmentioning

confidence: 99%

Human proinsulin production in the milk of transgenic cattle

Monzani,

Sangalli,

Sampaio

et al. 2024

Biotechnology Journal

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“…16 In contrast to the plasmid vectors, there are no commercial lentiviral vectors available for expressing recombinant proteins in milk, although some authors have modified commercial lentiviral vectors for this purpose 17,18,19 (Fig. 1).…”

Section: Vectors Used In the Generation Of Transgenic Animalsmentioning

confidence: 99%

“…29 Others studies have used MEC cultures to evaluate the expression of the vectors constructed. 13,18,19,30 Although transgenic mice may be the best model for transgenic farm animals, MEC culture is a strategy that is easier to execute than the generation of transgenic mice and shows certain advantages, such as a lower cost and an elimination of the necessity for a vivarium to produce and house transgenic mice.…”

Section: Vectors Used In the Generation Of Transgenic Animalsmentioning

confidence: 99%

Transgenic bovine as bioreactors: Challenges and perspectives

et al. 2016

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The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry.

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“…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 transduction. The analysis by q-PCR showed that the concentration of lentivirus from FUGW and the constructed vectors had similar quantities of lentiviral particles.…”

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Working with Lentivirus for Generation of Transgenic Bovine Embryos

Guemra¹

2014

Pharm Anal Acta

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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...

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MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Cited by 8 publications

References 24 publications

Human proinsulin production in the milk of transgenic cattle

Human proinsulin production in the milk of transgenic cattle

Transgenic bovine as bioreactors: Challenges and perspectives

Working with Lentivirus for Generation of Transgenic Bovine Embryos

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