Human proinsulin production in the milk of transgenic cattle

Background The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. Methods and Results Pseudo‐lentivirus containing the bovine β‐casein promoter and human insulin sequences was used to produce modified adult fibrob...

Full description

Saved in:
Bibliographic Details
Published in:Biotechnology journal 2024-03, Vol.19 (3), p.e2300307-n/a
Main Authors: Monzani, Paulo S., Sangalli, Juliano R., Sampaio, Rafael V., Guemra, Samuel, Zanin, Renato, Adona, Paulo R., Berlingieri, Maria A., Cunha‐Filho, Luiz F. C., Mora‐ Ocampo, Irma Y., Pirovani, Carlos P., Meirelles, Flávio V., Wheeler, Matthew B., Ohashi, Otavio M.
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified - metabolism
Cattle
cloning
Female
Humans
insulin
Insulin - analysis
lentivirus
Mac‐T cells
Milk - chemistry
milk mass spectrometry
Peptide Hydrolases - metabolism
Pregnancy
Proinsulin - analysis
Proinsulin - metabolism
Recombinant Proteins - metabolism
transgenic cow
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Background The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. Methods and Results Pseudo‐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. Conclusion The 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. Graphical and Lay Summary A lentiviral vector for the expression of human proinsulin driven by the B‐casein promoter was constructed and used for lentivirus production in HEK293FT cells. Lentivirus was used to transfect bovine fibroblasts, which were selected by blasticidin. Modified cells were used in somatic cell nuclear transfer to produce transgenic embryos. The embryos were transferred to recipient cows. A pregnancy was obtained, giving rise to the natural birth of a transgenic calf. In adulthood, lactation was induced, and the recombinant protein in milk was observed by mass spectrometry.
ISSN:1860-6768
1860-7314
DOI:10.1002/biot.202300307