The goat β‑casein/CMV chimeric promoter drives the expression of hLF in transgenic goats produced by cell transgene microinjection

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.

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Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells

Transgenic Research ◽

10.1007/s11248-012-9634-y ◽

2012 ◽

Vol 22 (1) ◽

pp. 131-142 ◽

Cited By ~ 17

Author(s):

Amir Amiri Yekta ◽

Azam Dalman ◽

Poopak Eftekhari-Yazdi ◽

Mohammad Hossein Sanati ◽

Abdol Hossein Shahverdi ◽

...

Keyword(s):

Nuclear Transfer ◽

Coagulation Factor ◽

Mammary Glands ◽

Factor Ix ◽

Fibroblast Cells ◽

Fetal Fibroblast ◽

Coagulation Factor Ix ◽

Transgenic Goats

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Laparoscopy vs. laparotomy for embryo transfer to produce transgenic goats (Capra hircus)

Journal of Veterinary Science ◽

10.4142/jvs.2008.9.1.103 ◽

2008 ◽

Vol 9 (1) ◽

pp. 103 ◽

Cited By ~ 8

Author(s):

Sang Tae Shin ◽

Sung Keun Jang ◽

Hong Suk Yang ◽

Ok Keun Lee ◽

Yhong Hee Shim ◽

...

Keyword(s):

Embryo Transfer ◽

Capra Hircus ◽

Transgenic Goats

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State of the art in the production of transgenic goats

Reproduction Fertility and Development ◽

10.1071/rd04028 ◽

2004 ◽

Vol 16 (4) ◽

pp. 465 ◽

Cited By ~ 30

Author(s):

H. Baldassarre ◽

B. Wang ◽

C. L. Keefer ◽

A. Lazaris ◽

C. N. Karatzas

Keyword(s):

Recombinant Proteins ◽

Traditional Method ◽

Nuclear Transfer ◽

State Of The Art ◽

Expression Vectors ◽

Marketing Approval ◽

Transfected Cells ◽

Pronuclear Microinjection ◽

Transgenic Goats

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

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Transgenic goats producing an improved version of cetuximab in milk

10.1101/2020.06.05.137463 ◽

2020 ◽

Author(s):

Götz Laible ◽

Sally Cole ◽

Brigid Brophy ◽

Paul Maclean ◽

Li How Chen ◽

...

Keyword(s):

Mammalian Cells ◽

Large Scale ◽

Growth Factor Receptor ◽

Cost Effective ◽

Scale Production ◽

Cancer Treatments ◽

Large Scale Production ◽

Immunogenic Epitope ◽

Dependent Cell ◽

Transgenic Goats

ABSTRACTTherapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

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Sequence-specific activation of transcription by adenovirus EIa products is observed in HeLa cells but not in 293 cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.201-208.1986 ◽

1986 ◽

Vol 6 (1) ◽

pp. 201-208

Author(s):

T Leff ◽

P Chambon

Keyword(s):

Hela Cells ◽

Upstream Region ◽

Gene Products ◽

Promoter Regions ◽

Chimeric Promoter ◽

293 Cells ◽

Activation Of Transcription ◽

Upstream Promoter ◽

Sequence Elements ◽

Responsive Element

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.

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Chimeric promoter

The Dictionary of Genomics, Transcriptomics and Proteomics ◽

10.1002/9783527678679.dg01650 ◽

2015 ◽

pp. 1-1

Keyword(s):

Chimeric Promoter

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Production of human lactoferrin in animal milk1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o11-088 ◽

2012 ◽

Vol 90 (3) ◽

pp. 513-519 ◽

Cited By ~ 15

Author(s):

I.L. Goldman ◽

S.G. Georgieva ◽

Ya.G. Gurskiy ◽

A.N. Krasnov ◽

A.V. Deykin ◽

...

Keyword(s):

Peer Review Process ◽

Micrococcus Luteus ◽

Chemical Properties ◽

Human Lactoferrin ◽

Natural Ligands ◽

Terminal Amino ◽

Transgenic Goats ◽

Genetic Constructs ◽

Physical And Chemical ◽

Circular Dichroic

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.

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