Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis.

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The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 μM for 6-24 h.

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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SummaryThis study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

The aim of this study was to evaluate the effects of mammary biopsy procedures on gland function of goats submitted to hormonal lactation. Ten female Caninde goats, at the age of around 3 years, were used, and lactation was induced by using estrogen, progesterone, and prednisolone. Ultrasonographic examinations of mammary gland, milking, and analysis of physicochemical properties of milk were performed immediately before (0 h) and 24, 48, and 72 h after each biopsy procedure. Surgical mammary gland biopsies were obtained at days 5 and 26 of the lactation. Even after drying initiation, all animals produced milk for five months. No abnormalities were observed on the ultrasonographic appearance of biopsied glands. All 20 biopsies were technically successful and any inflammation or infection was associated with biopsy procedures. Regarding the physicochemical properties and volumes of milk produced, no significant differences (P > 0.05) were observed between right and left glands nor in the comparison of the results from time 0 with 24, 48, and 72 h for biopsied teats. The mammary biopsy did not induce neither severe changes in production and physicochemical properties of the milk nor in ultrasonographic appearance of mammary gland of goats in hormonal lactation.Keywords: biopsy; goat; udder; ultrasonography. ResumoO objetivo deste estudo foi avaliar os efeitos dos procedimentos de biópsia mamária sobre a função da glândula de caprinos submetidos à lactação induzida. Dez cabras Canindé com idade de cerca de 3 anos foram utilizadas e a lactação foi induzida pelo uso de estrógeno, progesterona e prednisolona. Exames ultrassonográficos da glândula mamária, ordenha e análise das propriedades físico-químicas do leite foram realizados imediatamente antes (0 h) e 24, 48 e 72 h após cada procedimento de biópsia. As biópsias cirúrgicas de glândula mamária foram obtidas aos dias 5 e 26 da lactação. Mesmo após o início do processo de secagem, todos os animais continuaram produzindo leite por cinco meses. Nenhuma anormalidade foi observada na aparência ultrassonográfica das glândulas biopsiadas. Todas as 20 biópsias foram realizadas com sucesso e nenhuma inflamação ou infecção foi observada. Com relação às propriedades físico-químicas e volumes de leite produzido, nenhuma diferença significativa Cienc. anim. bras., Goiânia, v.18, 1-7, e-39077, 2017 2 SOUSA, F.C. et al.(P > 0,05) foi observada entre a glândula direita e esquerda, nem comparando resultados do momento 0 com 24, 48 e 72 h para as glândulas biopsiadas. A biópsia mamária não induz severas mudanças na produção e nas propriedades físico-químicas do leite nem na aparência ultrassonográfica da glândula mamária de caprinos em lactação.

SummaryThis study aimed to compare the efficiency of different incubation systems for in vitro embryo production in bovine. Oocytes/embryos were cultured in three incubators: conventional - CONV, mini bench - MINI and portable - PORT. After in vitro maturation (IVM), oocytes were verified for maturation rate. The remaining structures were submitted to in vitro fertilization and culture to verify cleavage (day 2) and blastocyst (day 7) rates. Reactive oxygen species (ROS) were evaluated in post-IVM oocytes and embryos (days 2 and 7) using arbitrary fluorescence units (AFUs). No significant difference (P>0.05) was observed for maturation rate. The CONV system (74.0%) produced the highest cleavage rate (P0.05) to MINI (65.0%). The same pattern and differences were observed for blastocyst rate: CONV (33.3%), MINI (32.3%) and PORT (21.9%). ROS levels were not different (P>0.05) in post-IVM oocytes: CONV (35.6±4.5), MINI (29.4±4.0) and PORT (35.6±4.5). For day-2 embryos, ROS levels were higher (P0.05) was observed in blastocysts. In conclusion, although it produced high ROS levels at day 2 of culture, the MINI system was as efficient as the CONV system for blastocyst production. This option may be an interesting and economical for the in vitro embryo industry.