β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

Monzani, Paulo Sérgio; Bressan, Fabiana Fernandes; Mesquita, Lígia Garcia; Sangalli, Juliano Rodrigues; Meirelles, Flávio Vieira

doi:10.4238/vol10-2gmr1034

Cited by 18 publications

(28 citation statements)

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“…Recently, Monzani et al (2011Monzani et al ( , 2013 reported the production of epithelial cell lineages derived from mammary gland expressing the recombinant human coagulation Factor IX (rFIX). The rFIX is an example of recombinant protein of biomedical interest which large-scale production would greatly benefit from bioreactor animals, once its expression and purification from in vitro cell cultures is expensive, and large-scale production is limited.…”

Section: Somatic Cell Nuclear Transfer As the Methods Of Choice For Thmentioning

confidence: 99%

Insights on bovine genetic engineering and cloning

et al. 2013

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Pesq. Vet. Bras. 33(Supl.1):113-118, dezembro 2013 113 RESUMO.-[Experiências em clonagem e transgenia em bovinos.] Tecnologias de modificação genética têm se tornado ferramentas essenciais para o desenvolvimento de biotecnologias animais. A clonagem animal mediante transferência nuclear de célula somática (TNCS) possibilitou a geração de animais geneticamente modificados através da utilização de linhagens celulares previamente modificadas e selecionadas como doadoras de núcleo, garantindo desta maneira a geração de rebanhos homogênoes expressando a modificação desejada. O presente estudo objetivou discutir o uso da TNCS como importante metodologia para a produção de rebanhos transgênicos, assim como experiências recentes na manipulação genética de células doadoras de núcleo e possíveis efeitos da indução gênica à pluripotência na TNCS.TERMOS DE INDEXAÇÃO: Pluripotência induzida, tecnologia transgênica, transferência nuclear de célula somática, bovino. INTRODUCTIONThe technology of genetic engineering plays an important role on both basic and applied research, becoming an essential tool for the understanding of the biology and development of animal biotechnology. Such technology presents a wide range of applications, such as the production of biopharmaceuticals, studies on gene expression and its regulation, the improvement of animal production, as, for example, production of herds resistant to specific diseases, and many other biomedical and medical purposes (Jaenisch et al. 1988, Houdebine 2005.Over the last decades several techniques were developed in order to produce genetically modified animals, and three of them became more popular due to their simplicity and positive results: exogenous pronuclear DNA microinjection in zygotes, injection of genetically modified embryonic stem (ES) cells into blastocysts, and, more recently, retrovirus mediated gene transfer (Gordon & Ruddle 1981, Hammer et al. 1985, Gossler et al. 1986, Hofmann et al. 2003, 2004. Injection of ES cells into blastocysts is the most popular methodology of genetic manipulation in mice, once homologous recombination of ES cells in mice is feasible, allowing the contribution of modified cells in the embryos and therefore obtaining transgenic animals after breeding (Misra & Duncan 2002 Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT. al . 2007), hindering its use on the production of transgenic herds.As an alternative to the non-feasibility of ES injection in blast...

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Section: Somatic Cell Nuclear Transfer As the Methods Of Choice For Thmentioning

confidence: 99%

Insights on bovine genetic engineering and cloning

et al. 2013

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“…All of the constructs produced viral particles that were used to transduce BMECs; the largest construct was approximately 13 kb, which includes the 9.5-kb to be packaged as vector. BMECs were isolated from the mammary gland of a pregnant B. taurus and analyzed for their capacity to differentiate into alveoli-like structures and β-casein expression (Monzani et al, 2011). These cells were used for lentiviral transduction; transgenic cells were selected by antibiotic resistance and hFIX transgene presence was verified by PCR ( Figure 1B).…”

Section: Construction Of Vectors and Transgenic Bmecsmentioning

confidence: 99%

“…BMECs were cultivated on 60-mm dishes to 30% confluence, when cells were transduced by incubation Table 1. Sequence primers used for construction vectors (Monzani et al, 2011).…”

Section: Bmec Transductionmentioning

confidence: 99%

“…The resultant amplicons were the expected sizes, suggesting Table 2. Primers and probes used in the qPCR assays (Monzani et al, 2011).…”

Section: Construction Of Vectors and Transgenic Bmecsmentioning

confidence: 99%

“…Primary cells were isolated from the mammary glands of Holstein cows (Bos taurus) in late pregnancy and cultured as previously described (Monzani et al, 2011). BMECs were cultivated on 60-mm dishes to 30% confluence, when cells were transduced by incubation Table 1.…”

Section: Bmec Transductionmentioning

confidence: 99%

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Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning

Monzani¹,

Sangalli²,

De³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392-and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

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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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β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

Cited by 18 publications

References 24 publications

Insights on bovine genetic engineering and cloning

Insights on bovine genetic engineering and cloning

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning

Human proinsulin production in the milk of transgenic cattle

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