High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow

Salamone, D.; Barañao, Lino; Santos, C.B.; Bussmann, Leonardo E.; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto D.; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; Gonzalez, Eliseo A.; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Cynthia de Freitas

doi:10.1016/j.jbiotec.2006.01.005

Cited by 71 publications

(59 citation statements)

References 7 publications

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“…To date, the delivery of foreign DNA into farm animals can be achieved by several methods, including pronuclear microinjection [10], sperm-mediated gene transfer [11,12], electroporation [13], somatic cell nuclear transfer employing genetically modified donor cells [14], and gene transfer mediated by retroviruses [15] or cationic molecules [16]. However, the efficiency of these techniques still remains low.…”

Section: Introductionmentioning

confidence: 99%

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Campelo

Canel

Bevacqua

et al. 2016

J Assist Reprod Genet

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Purpose Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes. Methods PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid. Results Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgeneexpressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05). Conclusions Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.

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Section: Introductionmentioning

confidence: 99%

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Campelo

Canel

Bevacqua

et al. 2016

J Assist Reprod Genet

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show abstract

“…Nevertheless, this method has many drawbacks: injected sequences randomly insert as concatemers, they have variable expression depending on the site of integration, and they generally result in production of mosaic animals [2,3]. Cloning techniques using genetically modified cells have also been employed for transgenic animal production and have resulted in transgenic cattle [4,5]. The main advantage of cloning with a modified cell line is that it allows targeted transgenesis [6].…”

Section: Introductionmentioning

confidence: 99%

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

et al. 2013

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a b s t r a c tAlthough transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/ 119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%,11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

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“…Lactation of the transgenic calf was hormonally induced at the age of 7 mo, essentially as described (44), except that hormones were injected s.c. WT samples of colostrum (provided by Jessica van Leeuwen, AgResearch, Hamilton, New Zealand), milk generated by hormonal induction (35), and natural milk were derived from cows of similar breed. The Western analysis was performed as described for mouse milk with a detection limit of 0.04 μg for bovine BLG.…”

Section: Methodsmentioning

confidence: 99%

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

Jabed

Wagner

McCracken

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA–mediated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits.

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High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow

Cited by 71 publications

References 7 publications

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

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