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 x 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.
Project description:Human lactoferrin (hLF) is a valuable protein for pharmaceutical products and functional foods, and worldwide demand for this protein has steadily increased. However, large-scale recombinant human lactoferrin (rhLF) production using current animal bioreactor techniques is limited by the low expression of foreign proteins, the use of antibiotic resistance genes and the down-regulation of endogenous milk proteins. Here, we generated a herd of marker-free, hLF bacterial artificial chromosome (BAC) transgenic cloned cows, as confirmed by Polymerase chain reaction, Southern blot and Western blot analyses. These transgenic cloned cows produced rhLF in milk at concentrations of 4.5-13.6?g/L. Moreover, the total protein content of the milk was increased. Over two hundred transgenic cloned cows were propagated by multiple ovulation and embryo transfer (MOET). A total of 400-450?g of rhLF protein, which shows similar enzymatic activity to natural hLF in iron binding and release, can be purified on a large scale from >100?L of milk per day. Our results suggested that transgenic bovine mammary bioreactors have the potential for large-scale protein production.
Project description:Background:Silk glands are used by silkworms to spin silk fibers for making their cocoons. These have recently been regarded as bioreactor hosts for the cost-effective production of other valuable exogenous proteins and have drawn wide attention. Results:In this study, we established a transgenic silkworm strain which synthesizes the recombinant human lactoferrin (rhLF) in the silk gland and spins them into the cocoon by our previously constructed silk gland based bioreactor system. The yield of the rhLF with the highest expression level was estimated to be 12.07?mg/g cocoon shell weight produced by the transgenic silkworm strain 34. Utilizing a simple purification protocol, 9.24?mg of the rhLF with recovery of 76.55% and purity of 95.45% on average could be purified from 1?g of the cocoons. The purified rhLF was detected with a secondary structure similar with the commercially purchased human lactoferrin. Eight types of N-glycans which dominated by the GlcNAc (4) Man (3) (61.15%) and the GlcNAc (3) Man (3) (17.98%) were identified at the three typical N-glycosylation sites of the rhLF. Biological activities assays showed the significant evidence that the purified rhLF could relief the lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells and exhibit potent antibacterial bioactivities against the Escherichia coli (E. coli) and Bacillus subtilis. Conclusions:These results show that the middle silk gland of silkworm can be an efficient bioreactor for the mass production of rhLF and the potential application in anti-inflammation and antibacterial.
Project description:There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce transgenic cloned goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing goat ??casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25?rhLF?Neo vector was microinjected into goat fetal fibroblasts. G418 selection and PCR analysis were used to identify transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this transgenic goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high?purity (95.8%) rhLF from a cation?exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a transgenic goat harboring the hLF transgene driven by the goat ??casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.
Project description:Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF.
Project description:This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.
Project description:Lactoferrin is a bioactive globular protein with unique properties towards musculo-skeletal cells and anabolic to bone in vivo. Even though the potent anti-apoptotic and osteogenic activity of lactoferrin has been reported, the mechanism of action has not been fully elucidated. The study demonstrates that the anti-apoptotic effect of rhLF towards MC3T3 pre-osteoblast cells is mediated by Wnt5a/PKA pathway and the stabilization of ?-catenin by rhLF is dependent on PKA/LRP6 signaling pathway. The study also investigates the feasibility of developing rhLF as a biomaterial for cell delivery. The injectable rhLF cell delivery vehicles are prepared by enzymatic crosslinking of tyramine-modified rhLF in the presence of hydrogen peroxide and horseradish peroxidase. The modified rhLF shows bioactivity similar to unmodified rhLF. The rhLF gels support encapsulated MC3T3 cell viability, proliferation, and differentiation, as well as phosphorylation of signaling proteins. In conclusion, the study demonstrates the involvement of Wnt5a, LRP6, and PKA signaling in rhLF-mediated bioactivity towards MC3T3 cells and the feasibility of developing an injectable cell delivery vehicle from rhLF.
Project description:Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P?<?0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.
Project description:Lactoferrin (LF), which belongs to the iron-binding transferrin family, is an important regulator of the levels of free iron in the body fluids. LF has raised significant interest as a bioactive protein due to its wide array of physiological effects on many different cell types, including osteoblasts and osteoclasts. The glycoprotein's degree of iron saturation has a pivotal influence on its physical structure. The objective of this study is to investigate the biological effects of apo (low iron saturation), pis (partially iron saturated), and holo (high iron saturation) recombinant human LF (rhLF) on MC3T3-E1 cells to identify the suitable candidate for bone tissue engineering application. Our studies demonstrated a dose-dependent mitogenic response of MC3T3 to rhLF treatment irrespective of the iron concentration. Furthermore, rhLF induced the cells to produce transcription factors, chemokines, and cytokines as determined by ?-catenin activation, phosphorylation of Akt, vascular endothelial growth factor, and interleukin (IL-6) expression. The iron saturation of rhLF did not have any significant effect on these biological activities of MC3T3 cells. In addition, the overall pattern of gene regulation in MC3T3-E1 cells upon rhLF treatment was followed by a global microarray analysis. Among the 45,200 genes tested, only 251 genes were found to be regulated by rhLFs of different iron concentrations. Of these, the transferrin receptor (Tfrc) was the only gene differentially regulated by the iron saturated and iron depleted (apo) rhLFs. In conclusion, the study demonstrated that rhLF is a bioactive protein and that the iron saturation of rhLF may not play a significant role in modulating osteoblast functions.
Project description:Lactoferrin (LF), an iron binding protein with immune modulatory activities, has adjuvant activity to enhance vaccine efficacy. Tuberculosis (TB) is a pulmonary disease caused by the pathogen Mycobacterium tuberculosis (MTB). Progressive TB disease is clinically defined by damaging pulmonary pathology, a result of inflammation due to immune reactivity. The current vaccine for TB, an attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), has only limited efficacy to prevent adult pulmonary TB. This study examines a Chinese hamster ovary (CHO) expressed recombinant human LF (rHLF) to boost efficacy of the BCG vaccine and delay early pathology post infectious challenge. C57BL/6 mice were immunized with BCG, or BCG admixed with either rHLF or bovine LF (bLF; internal control), or remained unvaccinated. Mice were then aerosol challenged with Erdman MTB. All vaccinated mice demonstrated decreased organ bacterial load up to 19 weeks post infection compared with non-vaccinated controls. Furthermore, mice receiving bLF or rHLF supplemented BCG vaccines showed a modest decrease in lung pathology developed over time, compared to the BCG vaccine alone. While mice vaccinated with BCG/rHLF demonstrated increased general lung inflammation at day 7, it occurred without noticeable increase in pro-inflammatory cytokines. At later times, decreased pathology in the rHLF groups correlated with decreased inflammatory cytokines. Splenic recall to BCG antigens showed BCG/rHLF vaccination increased production of IFN-?, IL-6, and GM-CSF compared to naïve, BCG, and BCG/bLF groups. Analysis of T cell stimulating functions of bone marrow derived macrophages and dendritic cells treated with BCG/bLF or BCG/rHLF showed decreases in IL-10 production when co-cultured with sensitized CD4 and CD8 T cells, compared to those cultured with macrophages/dendritic cells treated with BCG without LF. These results indicate that addition of rHLF to the BCG vaccine can modulate development of host pathology early post infectious challenge, most likely through host immune regulation affecting hypersensitive responses.
Project description:We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg2-Arg3-Arg4-Arg5 of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf-2N, hLf-3N and hLf-4N) from native hLf that had been treated with trypsin. Native hLf bound to 102000 sites on Jurkat cells with a dissociation constant (Kd) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf-5N) bound to 17000 sites with a Kd of 12 nM. The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; Kd 30 nM) resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N, indicating that the hLf-binding sites include sulphated molecules. We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg2-Arg3-Arg4, but not on Arg5. Interaction with approx. 20000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. Moreover, the affinity of hLf for the latter binding site is enhanced approx. 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.