The establishment of two transgenic goat lines for mammary gland hG-CSF expression

Freitas, V. J. F.; Serova, Irina; Moura, R.R.; Andreeva, L. E.; Melo, Luciana Magalhães; Teixeira, Dárcio Ítalo Alves; Pereira, Alexsandra Fernandes; Lopes-Jr, E.S.; Dias, L.P.B.; Nunes-Pinheiro, Diana Célia Sousa; Sousa, Francisco Carlos de; Alcântara-Neto, A.S.; Albuquerque, Erica S.; Melo, Cynthia de Freitas; Rodrigues, Valeska; Batista, Ríbrio Ivan Tavares Pereira; Dvoryanchikov, Gennady; Serov, O. L.

doi:10.1016/j.smallrumres.2012.03.009

Cited by 14 publications

(15 citation statements)

References 25 publications

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“…DNA samples were eluted or resuspended in 100 μL nuclease‐free water and stored at −80°C until qPCR. Plasmid DNA was obtained as described previously . DNA concentrations were accurately accessed by fluorimetry using the Qubit fluorimeter (Invitrogen) and Quant‐iT dsDNA BR Assay Kit (Invitrogen) for gDNA or Quant‐iT dsDNA HS Assay Kit (Invitrogen) for plasmid DNA following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid DNA was obtained as described previously. 6 DNA concentrations were accurately accessed by fluorimetry using the Qubit fluorimeter (Invitrogen) and Quant-iT dsDNA BR Assay Kit (Invitrogen) for gDNA or Quant-iT dsDNA HS Assay Kit (Invitrogen) for plasmid DNA following the manufacturer's instructions. Derivative melting curves for both TgCD and Gluc amplicons were plotted.…”

Section: Gdna Extractionmentioning

confidence: 99%

“…1 In this context, due to the equilibrium between the maintenance cost, milk production and precocity, goats (Capra hircus) became one of the first choices of a model for animal transgenesis, 2 as indicated by the variety of human recombinant proteins produced by the mammary gland and secreted in the milk of transgenic goats, such as antithrombin, 3 lysozyme, 4 lactoferrin, 5 and granulocyte-colony stimulating factor (hG-CSF). 6 In addition, the European Medicines Agency announced approval of the first drug produced in an animal bioreactor: GTC Biotherapeutics' ATrynV R (recombinant human antithrombin). 7 Traditional techniques used to produce transgenic goats randomly integrate the foreign gene into a chromosome of the founder, usually in multiple copies.…”

Section: Introductionmentioning

confidence: 99%

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Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Batista

Luciano

Teixeira

et al. 2014

Biotechnology Progress

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Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

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Section: Methodsmentioning

confidence: 99%

Section: Gdna Extractionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Batista

Luciano

Teixeira

et al. 2014

Biotechnology Progress

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“…The experimental animals were six Canindé male goats, two of which were transgenic (T) for hG-CSF milk expression (F1 specimens of the 10M line described by Freitas et al, 2012) and four were non-transgenic (NT). The bucks aged 2-3 years old and showed body weights of 41.2 ± 0.9 kg and a body condition score of 3.0 ± 0.6 (1-5 scale).…”

Section: Animals and Managementmentioning

confidence: 99%

“…Therefore, the aim of the current study was to compare LP and US-guided biopsy methods to collect reliable liver and splenic tissue samples for gene expression analysis in transgenic goats. First generation (F1) male bucks of a transgenic line for human granulocyte colony-stimulating factor (hG-CSF) milk expression previously produced by our group (Freitas et al, 2012) were used.…”

Section: Introductionmentioning

confidence: 99%

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats

et al. 2015

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ABSTRACT. The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to realtime PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver 8673 Biopsy methods for ectopic expression in transgenic bucks ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 8672-8684 (2015) samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.

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Reproductive Biotechnologies Applied to the Female Sheep and Goat

Souza-Fabjan

Alves

Batista

et al. 2023

Sustainable Agriculture Reviews

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The establishment of two transgenic goat lines for mammary gland hG-CSF expression

Cited by 14 publications

References 25 publications

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats

Reproductive Biotechnologies Applied to the Female Sheep and Goat

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