A novel recombinant human plasminogen activator: Efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats

Abstract

BackgroundThromboses is a rapidly growing medical problem worldwide. Low-cost, high-scale production of thrombotic drugs is needed to meet the demand. The production of biomolecules in transgenic animals might help address this issue. To our knowledge, the expression of recombinant human plasminogen activator (rhPA) in goat mammary glands has never been reported before.MethodsWe constructed a mammary gland–specific expression vector, BLC14/rhPA, which encodes only the essential K2 fibrin-binding and P domains of wild-type tPA (deletion mutant of tPA lacking the F, E, and K1 domains), along with the goat β-lactoglobulin gene signal peptide-coding sequence. The mammary gland–specific expression vector BLC14/rhPA was transfected into goat fetal fibroblast cells by electroporation. After selection for 3 weeks by G418, stably transfected cell colonies were obtained. PCR analysis results indicated that 24 of the resistant clones were transgenic cell lines; of these, 8 lines were selected as the donor cells. The positive cells were starved for 72 h with DMEM/F12 medium containing 0.5% FBS and were then used as do. Finally, 256 reconstructed oocytes were transferred into 26 recipients, and 7 of them became pregnant (pregnancy rate, 26.9%). Two kids were obtained (BP21 and BP22). PCR analysis confirmed that both were transgenic goats. To analyze the heredity of the rhPA expressed in BP21 F0 and F1 transgenic goats, the F0 transgenic goat BP21 was mated with a normal male goat to generate an F1 transgenic goat. Enucleated metaphase II (MII) oocytes and positive donor cells were used to reconstruct embryos, which were transplanted into the oviducts of the recipients.ResultsWestern blot results showed a specific 39 kDa band. The rhPA expression level in transgenic goat whey was about 78.32 μg/mL by ELISA. Results of ELISA and the in vitro thrombolysis test (FAPA) showed that specific activity of the rhPA in the milk of F0 and F1 transgenic goats was 13.3 times higher than that of the reteplase reference material.ConclusionThus, we demonstrated that BLC14/rhPA was reasonably effective for expression in the mammary glands of transgenic goats, and was stably inherited by the offspring. This study provides the basis for the large-scale production of biological pharmaceuticals in transgenic animals. The expression of biopharmaceuticals by transgenic animals can be used for pharmacological research and bioactive analysis, and transgenic goats were demonstrated to be promising animals for the large-scale production of thrombolytic biopharmaceuticals.

Production of Bioactive Recombinant Reteplase by Virus-Based Transient Expression System in Nicotiana benthamiana

“…This high pricing has limited the application of the recombinant rPA, especially in the developing and third-world countries. To overcome this issue, alternative production systems have been tested: Chinese hamster ovary cells (Davami et al, 2010), insect cells (Aflakiyan et al, 2013), yeast (Shu-Guang et al, 2006), transgenic animals (He et al, 2018), and transgenic plants (Zhang et al, 2008; Nasab et al, 2016; Hidalgo et al, 2017). However, most of these were not found to be appropriate.…”

Developmental analysis of reconstructed embryos of second‐generation cloned transgenic goats

“…These two medical proteins play an important role in clinic and have been widely studied and applied by scientists at home and abroad. Our team has previously produced tPA and LTF transgenic cloned goats with high expression levels using SCNT and successfully obtained these two foreign proteins with superior performance (He et al., 2018; Yuan et al., 2017). Therefore, choosing these two valuable gene expression vectors as research objects has a certain research foundation.…”

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals

“…Animal cloning by somatic cell nuclear transfer (SCNT), which avoids the sexual reproduction pathway, offers the opportunity to obtain monogenetic offspring derived not only from adult animals of high genetic merit but also from genetically transformed (transgenic) specimens. Over the last 24 years, intra- and interspecies cloning via SCNT resulted in a fairly large number of transgenic and non-transgenic offspring, not only in various species or infertile interspecific hybrids (bastards) of domesticated animals, such as cattle [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]; goats [ 6 , 15 , 16 , 17 , 18 , 19 , 20 ]; sheep [ 21 , 22 , 23 , 24 , 25 , 26 ]; pigs [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ]; equids—domestic horses [ 42 , 43 , 44 , 45 , 46 ] and mules [ 47 ]; water buffaloes—Chinese swamp buffaloes [ 48 , 49 ] and Indian river/riverine buffaloes [ 50 , ...…”