Introduction
Transgenic technology in animals has been extensively used for many purposes. The use of the traditional methods to obtain transgenic mammals dates back to the 1970s (Jaenisch and Mintz, Reference Jaenisch and Mintz1974) and led to the production of the first transgenic mice by pronuclear microinjection (Gordon et al., Reference Gordon, Scangos, Plotkin, Barbosa and Ruddle1980). Later, transgenic animals were produced from different species such as rabbit, sheep, pig (Hammer and Pursel, Reference Hammer and Pursel1985), cattle (Krimpenfort et al., Reference Krimpenfort, Rademakers, Eyestone, van der Schans, van den Broek, Kooiman, Kootwijk, Platenburg, Pieper, Strijker and de Boer1991) and goat (Ebert et al., Reference Ebert, Selgrath, DiTullio, Denman, Smith, Memon, Schindler, Monastersky, Vitale and Gordon1991). However, this technique presents several drawbacks, ranging from low transfection efficiency to high cost of equipment and the need for skilled personal (Kues and Niemann, Reference Kues and Niemann2011). To overcome these disadvantages several strategies have been developed such as sperm-mediated gene transfer (Chang et al., Reference Chang, Qian, Jiang, Liu, Wu, Chen, Lai, Lo, Hsiao, Brown, Bolen, Huang, Ho, Shih, Yao, Lin, Chen, Wu, Lin, Xu and Wang2002), gene transfer mediated by cationic lipids (Carballada et al., Reference Carballada, Relloso and Esponda2002), transfection of donor cells followed by nuclear transfer (Salamone et al., Reference Salamone, Barañao, Santos, Bussmann, Artuso, Werning, Prync, Carbonetto, Dabsys, Munar, Salaberry, Berra, Berra, Fernández, Papouchado, Foti, Judewicz, Mujica, Muñoz, Alvarez, González, Zimmermann, Criscuolo and Melo2006), gene transfer mediated by retroviruses (Xu et al., Reference Xu, Uhm, Koo, Kwon, Roh, Yang, Choi, Heo, Cui, Yoon, Ko, Kim and Kim2013) and, more recently, genome editing using the CRISPR/Cas9 system (Li et al., Reference Li, Miao, Fan, Song, Kong, Wang and Wang2018).
For continuous improvement of transgenic technology, it is necessary to develop simple, affordable and efficient methods. Therefore, in recent decades, studies concerning the use of cell-penetrating peptides (CPPs) in transgenesis have been investigated (Rádis-Baptista et al., Reference Rádis-Baptista, Campelo, Morlighem, Melo and Freitas2017). CPPs are represented by short cationic or amphipathic oligopeptides that translocate across the plasma membrane and penetrate eukaryotic cells (Pooga and Langel, Reference Pooga and Langel2015). These peptides can usually carry different categories of therapeutic substances into the cells, including pharmaceuticals, fluorescent probes, proteins, DNA and RNA (Stewart et al., Reference Stewart, Horton and Kelley2008).
A few years ago, our group started studies with crotamine, one of these CPPs, for potential use in animal transgenesis (Campelo et al., Reference Campelo, Pereira, Alcântara-Neto, Canel, Souza-Fabjan, Teixeira, Camargo, Melo, Rádis-Baptista, Salamone and Freitas2016a, Reference Campelo, Canel, Bevacqua, Melo, Rádis-Baptista, Freitas and Salamone2016b), exploring its ability to stable binding to nucleic acids by electrostatic interaction and forming DNA–peptide complex. Crotamine was first reported back in 1947 as one of the predominant components in the venom of South American rattlesnake Crotalus durissus terrificus (Gonçalves and Polson, Reference Gonçalves and Polson1947). More recently, crotamine was described as a novel CPP capable of translocation and be located in the cytoplasm of different eukaryotic cells (Kerkis et al., Reference Kerkis, Kerkis, Rádis-Baptista, Oliveira, Vianna-Morgante, Pereira and Yamane2004; Nascimento et al., Reference Nascimento, Hayashi, Kerkis, Oliveira, Oliveira, Rádis-Baptista, Nader, Yamane, Tersariol and Kerkis2007) and preimplantation embryos such as mouse morulas (Kerkis et al., Reference Kerkis, Kerkis, Rádis-Baptista, Oliveira, Vianna-Morgante, Pereira and Yamane2004) and bovine zygotes (Campelo et al., Reference Campelo, Pereira, Alcântara-Neto, Canel, Souza-Fabjan, Teixeira, Camargo, Melo, Rádis-Baptista, Salamone and Freitas2016a). It was also demonstrated that this venom-derived peptide is able to successfully transfect cells in murine model (Nascimento et al., Reference Nascimento, Hayashi, Kerkis, Oliveira, Oliveira, Rádis-Baptista, Nader, Yamane, Tersariol and Kerkis2007). However, when using bovine as a model for gene delivery, crotamine did not improve gene transfer possibly due to its high affinity for DNA molecules (Campelo et al., Reference Campelo, Canel, Bevacqua, Melo, Rádis-Baptista, Freitas and Salamone2016b).
Therefore, crotamine structural derivatives and analogues have been designed and investigated for their efficiency of cell penetration and delivery of molecules. In this scenario, short crotamine-derived peptides (named NrTPs) have been evaluated for cellular uptake and retained some desirable properties of crotamine such as efficient cell uptake, nuclear homing and cellular labelling. Noteworthy, other advantageous properties (short size, nucleolar targeting, facility for zip-code localization of therapeutic protein) were improved in comparison with the native peptide (Rádis-Baptista et al., Reference Rádis-Baptista, de la Torre and Andreu2008; Rádis-Baptista et al., Reference Rádis-Baptista, de la Torre and Andreu2012). Therefore, the aim of the present study was to investigate the feasibility of using another variation of crotamine structure, i.e. a disulphide-less (linear) crotamine (dLCr) that could deliver nucleic acids (genes) and facilitate the DNA–peptide intracellular dissociation, aimed at the transfection of bovine embryos. With such purpose, the present work evaluated: (i) the ability of dLCr to form complex, under several in vitro conditions, with a plasmid harbouring the reporter gene for green fluorescent protein (GFP); (ii) the toxicity of dLCr toward in vitro fertilized (IVF) bovine embryos; and (iii) the effectiveness of dLCr to improve the expression of GFP transgene in embryos derived from in vitro fertilized bovine zygotes injected with DNA–dLCr.
Material and methods
Experimental design
This study consisted of three different experiments. In experiment 1, DNA–dLCr complex formation and stability were initially evaluated under different in vitro conditions to establish the parameters for further embryo transfection. In experiment 2, dLCr embryo toxicity was assessed by exposure of bovine embryos to dLCr. Finally, in experiment 3, expression of the GFP reporter gene was used to assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or GFP plasmid alone (control).
Disulphide-less and native versions of crotamine
Native crotamine (NCr) was purified from the crude venom of rattlesnakes kept in the serpentarium of São Paulo University (Ribeirão Preto, Brazil). The method for NCr preparation was as described previously (Kerkis et al., Reference Kerkis, Kerkis, Rádis-Baptista, Oliveira, Vianna-Morgante, Pereira and Yamane2004). dLCr was prepared commercially by solid peptide synthesis (China Peptides Co., Shanghai, China) using the designed sequence C-YKQSHKKGGHAbuFPKEKIAbuLPPSSDFGKMDAbuRWRWKAbuAbuKKGSG (MW 4886.73). This new version of crotamine structure does not form intrachain or interchain disulphide bonds due to the replacement of cysteine residues for 2-aminobutyric acid (Abu). Lyophilized dLCr was stored at −20°C until use.
Plasmid DNA preparation
The circular plasmid pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA), comprised of a reporter gene encoding GFP driven by the cytomegalovirus (CMV) promoter (GenBank accession no. U55762) was used. The plasmid was cloned and propagated in Escherichia coli and purified using the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer’s instructions.
Experiment 1: Conditions of DNA–dLCr complex formation
Stoichiometry and kinetics of complex formation
To determine the stoichiometry of DNA–dLCr complex, a fixed concentration of pEGFP-N1 (25 ng/µl) was combined with increasing concentrations of dLCr (0.25, 0.625, 1.25 or 2.5 µg/µl) resulting in DNA:peptide mass ratios of 1:10, 1:25, 1:50 and 1:100, respectively. Both DNA–NCr complex and DNA-lipofectamine lipoplex were positive controls. A DNA–NCr complex was formed by mixing pEGFP-N1 at 25 ng/µl with NCr at 0.25 µg/µl (1:10; DNA:peptide; wt:wt). The lipoplex was prepared using Lipofectamine 2000 Reagent (Invitrogen Co.) and pEGFP-N1 as indicated by the manufacturer. All complex formations were measured after 30 min of incubation. In another assay, the kinetics of complex formation was monitored at 0, 5, 30, 60 and 90 min. DNA–peptides complexes were prepared as described above to achieve DNA:peptide mass ratios of 1:10, 1:25 and 1:50 for DNA–dLCr, and 1:10 for the DNA–NCr (positive control). For both assays, the complexes were prepared in 150 mM NaCl solution at approximately 26°C. The percentage of complexed DNA was measured indirectly using an ‘in-lab’ developed methodology based on the fluorophore exclusion assay and the Qubit dsDNA High Sensitivity (HS) Assay Kit (Life Technologies, Eugene, OR, USA), using a Qubit 2.0 fluorimeter (Invitrogen Co.) for readings, as previously described (Freitas et al., Reference Freitas, Alcântara-Neto, Pereira, Campelo, Melo and Rádis-Baptista2014). Negative controls were prepared using DNA at the same concentrations described previously in the absence of peptide and were used for fluorescence signal normalization. Each assay was repeated three times and groups were performed in duplicate within repetitions.
Complex stability in embryo culture medium
The stability of the DNA–dLCr complex was evaluated in synthetic oviductal fluid (SOF) medium in the absence of fetal calf serum, albumin and amino acids (SOF-saa). Therefore, the DNA–dLCr complex was previously prepared by 30 min incubation of dLCr mixed with pEGFP-N1 at a DNA:peptide mass ratio of 1:25 (the best condition defined in the kinetics assay). Samples were recovered at 0, 180 and 360 min after DNA–dLCr dilution in SOF-saa medium (1:10; v:v) to measure the fluorescence signal. The concentration of complexed DNA was evaluated using the Qubit dsDNA HS Assay Kit and a Qubit 2.0 fluorimeter as previously. NCr was used as the positive control at a 1:10 DNA:peptide mass ratio as described before. The experimental groups were performed in duplicate in each repetition and the assay was repeated three times.
Experiment 2: Evaluation of dLCr embryotoxicity
Oocyte collection and in vitro maturation (IVM)
Bovine ovaries were collected from a local slaughterhouse and transported on ice to the laboratory in 0.9% (w:v) NaCl solution containing antibiotics (Pentabiótico; Fort Dodge, Campinas, Brazil). Cumulus–oocyte complexes (COCs) from follicles of 2–8 mm in diameter were aspirated using an 18-gauge needle attached to a 10 ml syringe. Only COCs with homogeneous cytoplasm surrounded by at least three layers of compact cumulus cells were selected for further IVM. After collection, the COCs were washed in manipulation medium composed of TCM-199 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% (v:v) antibiotic–antimycotic solution (Sigma-Aldrich), 0.1 mM sodium pyruvate (Sigma-Aldrich), 10% (v:v) fetal calf serum (FCS; Gibco, Grand Island, NY, USA) and 25 mM HEPES (Sigma-Aldrich). Groups of up to 50 COCs were placed in 500 µl of maturation medium composed of TCM-199 containing 10% (v:v) FCS, 0.1 mM sodium pyruvate, 10 ng/ml epidermal growth factor (Sigma-Aldrich), 100 μM cysteamine (Sigma-Aldrich), 20 μg/ml FSH/LH (Pluset; Hertape Calier, Barcelona, Spain), 1 μg/ml 17β-estradiol (Sigma-Aldrich), 1% (v:v) antibiotic–antimycotic solution, and 1 mM l-glutamine (Sigma-Aldrich). IVM was performed at 38.5°C for 23–24 h in an humidified atmosphere of 5% CO2.
In vitro fertilization (IVF)
Oocytes were submitted to IVF with frozen–thawed semen of a bull with proven fertility. The semen was thawed for 5 s in air and 25 s in a water bath at 37°C. The semen was separated in 4 ml of Percoll gradient (Sigma-Aldrich; 55%/90%) by centrifugation at 700 g for 25 min. The Percoll-separated semen was washed twice with 2 ml of BO medium added with 5 mM caffeine (Sigma-Aldrich) and 20 UI/ml heparin (Calbiochem, Merck, Darmstadt, Germany). After washing, the semen was diluted with BO added with 10 mg/ml bovine serum albumin (BSA; Sigma-Aldrich) to achieve a final concentration of 20 × 106 spermatozoa/ml. Finally, the COCs subjected to IVM were washed in manipulation medium and co-incubated with a suspension of semen in droplets of 100 µl covered with mineral oil (Sigma-Aldrich) at 38.5°C in a humidified atmosphere of 5% CO2. At 6 h post-IVF, presumptive zygotes were denuded by vortexing in manipulation medium and distributed randomly for respective groups of exposure or microinjection experiments.
Exposure to dLCr
Zygotes were exposed to dLCr for 6 h and after that they were transferred to droplets composed of 5 μl of dLCr solution and 45 μl of SOF-saa and maintained at 38.5°C in an atmosphere of 5% CO2, 5% O2, 90% N2. dLCr solution was prepared in NaCl 150 mM to achieve concentrations of 0 µM (vehicle), 5 µM (0.025 µg/µl) and 12.5 µM (0.0625 µg/µl) in the culture medium. After exposure, the embryos were washed extensively in manipulation medium and subjected to embryo culture as described ahead. A group not exposed to dLCr (IVF group) was performed as the negative control. In addition, the exposure of zygotes to higher concentrations of dLCr: 25 µM (0.125 µg/µl) and 50 µM (0.25 µg/µl) was evaluated.
Experiment 3: Assess the efficiency of bovine embryo transfection with DNA–dLCr
Cytoplasmic microinjection of DNA–dLCr complex
To assess the ability of dLCr to improve exogenous DNA expression, DNA–dLCr complexes were first microinjected into the cytoplasm of IVF presumptive zygotes. The complex was prepared with pEGFP-N1, which encodes a red shifted variant of the wild type GFP reporter gene, and dLCr in 150 mM NaCl for at least 30 min. The DNA:peptide mass ratio for complex formation was 1:25 (as defined in experiment 1). Also, a positive control group was performed by microinjection of a solution composed of only pEGFP-N1 plasmid (25 ng/µl). Next, 5 μl of solution were backfilled in microinjection capillaries (FemtoTip II; Eppendorf, Hamburg, Germany) with an internal tip diameter of approximately 0.5 μm. Presumptive zygotes were transferred to 50 μl droplets of manipulation medium in a 60 mm dish and covered with mineral oil. Zygotes were fixed by suction to a holding pipette, meanwhile the capillary was pushed through the embryo’s zona pellucida and cytoplasm membrane. DNA–dLCr and DNA alone solutions were injected using a FemtoJet (Eppendorf) set as follows: injection pressure = 300 hPa, compensation pressure = 10 hPa, injection time = 0.5 s. Embryo micromanipulation was performed in a Narishige hydraulic micromanipulator (Narishige Science, Tokyo, Japan) mounted on a Nikon Eclipse TE2000 inverted microscope (Nikon, Tokyo, Japan). The microinjection procedure is described in Supporting Information. After injection, the zygotes were transferred into droplets of culture medium. In addition, an IVF control group (not microinjected) was performed.
Embryo culture and in vitro development assessment
Embryo culture was performed in 50 μl droplets of SOF medium supplemented with 0.8% (w:v) BSA and 2.5% (v:v) FCS under mineral oil at 5% CO2, 5% O2, 90% N2 and 38.5°C in high humidity. Embryo cleavage rate was assessed at day 2 post-IVF and blastocyst formation rates were evaluated at day 7 (D7) and day 8 (D8). At D8, hatching/hatched embryos were counted to assess embryo quality by means of hatching rate. GFP expression was evaluated under ultraviolet (UV) light using the Nikon Eclipse TE2000 microscope (excitation filter at 488 nm and emission filter at 530 nm) at day 5 (D5) and D7 of in vitro culture.
Data and statistical analysis
Statistical analyses were performed using Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA). For stoichiometry assay, complexed DNA rate was assessed by one-way analysis of variance (ANOVA) multiple comparisons. In the kinetics assay, complexed DNA rate was evaluated by multiple t-test for differences between groups at the same time and by one-way ANOVA repeated measurements for differences between times at the same group. In complex stability assay, the complexed DNA rate was assessed by multiple t-test for differences between groups at the same time and by two-way ANOVA multiple comparisons for differences between times at the same group. The statistical analysis of cleavage, blastocyst, hatching and GFP expression rates was assessed using non-parametric Fisher’s exact test. For all tests, a P-value < 0.05 was considered to be statistically significant.
Results
Experiment 1: Stoichiometry and kinetics of complex formation
Figure 1 shows that proportions of 1:100 and 1:50 DNA–dLCr reached higher (P < 0.05) complexation efficiency than DNA–NCr 1:10 and lipofectamine. In addition, similar rates of complexed DNA (P > 0.05) were observed between those proportions, which were able to complex the maximal amount of DNA (over 90%) within 30 min of incubation. However, there was no difference when 1:25 DNA–dLCr and both controls (P > 0.05) were compared. Conversely, the proportion of 1:10 DNA–dLCr, resulted in the lowest (P < 0.05) complexation (less than 35%).
Figure 1. Stoichiometry of DNA–disulphide-less crotamine (dLCr) complex formation assessed by fluorophore exclusion assay. A DNA–dLCr complex was formed at 1:100, 1:50, 1:25, and 1:10 DNA:peptide mass ratios. Native crotamine (NCr) at 1:10 (DNA:peptide; wt:wt) and lipofectamine 2000 (Lipo) were used as positive controls. a–cDifferent lowercase letters indicate statistical differences (P < 0.05).
According to kinetics data, the DNA complexation by dLCr seems to occur very fast (Fig. 2). Therefore, for 1:50 or 1:25 DNA–dLCr more than 75% of the DNA was complexed by the peptide within 5 min of incubation. For all DNA–dLCr groups, the efficiency of the complexation was higher or similar than the 1:10 DNA–NCr group within 30 min of incubation. At 60 min incubation, the 1:50 DNA–dLCr group achieved a complexation rate of 99.6%, which was similar to 1:25 (95.8%) group, although higher (P < 0.05) than the 1:10 DNA–dLCr (63.0%) and 1:10 DNA–NCr (76.1%) groups. The 1:10 dLCr group seems to have the slowest complexation, reaching less than 25% DNA complexation at 5 min and less than 65% at 60 min.
Figure 2. Kinetics of DNA–disulfide-less crotamine (dLCr) complex formation assessed by fluorophore exclusion assay. A DNA–dLCr complex was formed at 1:50, 1:25, and 1:10 (DNA:peptide; wt:wt). Complexed DNA was measured at 0, 5, 30, 60 and 90 min of complexation. Native crotamine (NCr) was used as a positive control at 1:10 DNA:peptide mass ratio. Different lowercase letters (a–d) indicate statistical differences between groups at different times and different uppercase letters (A–C) indicate statistical differences between times within the same group (P < 0.05).
Experiment 1: Complex stability in embryo culture medium
To evaluate the stability of DNA–dLCr complexes in a DNA-free culture medium, complexes were incubated in SOF-saa medium and analyzed at 0, 180 and 360 min intervals (Fig. 3). No statistical difference was observed when comparing DNA–dLCr and DNA–NCr at the same time of incubation nor when comparing the different times within the same group (P > 0.05). Therefore, it was observed that there was no additional complexation or degradation of complexes previously formed in NaCl solution.
Figure 3. Stability of DNA–disulphide-less crotamine (dLCr) complex in embryo culture medium (SOF-saa) assessed by fluorophore exclusion assay. DNA–dLCr complex were formed at 1:25 DNA:peptide mass ratio for 30 min, diluted in SOF-saa, and then measured at 0, 180 and 360 min of incubation. Native crotamine (NCr) was used as positive control at 1:10 DNA:peptide mass ratio. There was no statistical difference between groups at diverse times and between times within the same group (P > 0.05).
Experiment 2: In vitro development of embryos exposed to dLCr
In this experiment, the potential embryotoxicity of dLCr was assessed by evaluating the in vitro development of IVF bovine embryos exposed to dLCr for 6 h. The effects were recorded by means of embryo cleavage rate, blastocyst formation at D7 and D8 rates and hatching rate of groups exposed to 5 or 12.5 µM dLCr. One control group consisted of embryos exposed to SOF-saa medium with vehicle solution and without dLCr for 6 h (0 µM dLCr), and another consisted of embryos not exposed to vehicle solution and dLCr (IVF control). In vitro embryo development is shown in Table 1.
Table 1. Effect of low concentrations of disulphide-less crotamine (dLCr) on development of in vitro fertilized (IVF) bovine embryos
Experiment was replicated four times.
a Similar superscripts within the same column mean statistical similarity (P < 0.05).
* Calculated from total number of blastocysts at day 8.
Cleavage, blastocyst productions and blastocyst hatching rates were similar among groups exposed to dLCr, 0 µM dLCr and IVF control (P > 0.05). In addition, higher dLCr concentrations (25 and 50 µM, i.e., 0.125 µg/µl and 0.25 µg/µl, respectively, for 6 h) also did not affect the development of in vitro fertilized bovine embryos (Table 2). Therefore, exposure of zygotes for 6 h to up to 50 µM dLCr had no detrimental effect on in vitro embryo development.
Table 2. Effect of high concentrations of disulphide-less crotamine (dLCr) on development of in vitro fertilized (IVF) bovine embryos
Experiment was replicated four times.
a,bDifferent superscripts within the same column differ significantly (P < 0.05).
* Calculated from total number of blastocysts.
Experiment 3: Transfection of embryos by microinjection of DNA–dLCr complex
Microinjection of the 1:25 DNA–dLCr complex was compared with microinjection of DNA alone by means of in vitro development and emission of GFP fluorescence. Also, an IVF control group was performed to assess the viability of in vitro embryo culture (Table 3). For embryo cleavage and blastocyst production (D7 and D8), microinjected groups had lower rates (P < 0.05) than the IVF control. Nevertheless, embryo development parameters were similar (P > 0.05) among groups of embryos microinjected (DNA–dLCr and DNA alone). To determine the quality of blastocysts formed, hatching rate was evaluated for all groups and no difference (P > 0.05) was found among these (Table 3).
Table 3. Embryo development and enhanced green fluorescent protein (EGFP) expression of microinjected in vitro fertilized (IVF) bovine embryos with DNA-disulphide-less crotamine (DLCr) complex (+) or plasmid DNA alone (−)
Experiment was replicated five times.
NA, not applicable.
*Rates calculated over zygotes. **Rates calculated from total number of blastocysts at day 8. ***Rates calculated over cleaved embryos.
a,bDifferent superscripts within the same column differ significantly (P < 0.05).
The rate of GFP expression is shown in Table 3. In total, 4.6% and 2.8% of cleaved embryos was observed to express fluorescence at D5 and D7, respectively, after microinjection of plasmid alone. Use of the DNA–dLCr complex for microinjection did not increase the number of GFP-positive embryos when compared with plasmid DNA alone (P > 0.05). Additionally, there was no detectable qualitative transgene expression on embryos microinjected with DNA–dLCr complex in comparison with microinjection of DNA plasmid alone.
Discussion
To date, this is the first study to describe the use of a synthetic version of disulphide-less (linear) crotamine for DNA complex formation and gene transfer. The native peptide is rich in positive amino acid residues (nine lysine and two arginine residues) and six cysteine. The positive residues conferred a net positive charge to this molecule, i.e. a basic characteristic and cysteine make up three precisely arranged disulphide bonds (Nicastro et al., Reference Nicastro, Franzoni, de Chiara, Mancin, Giglio and Spisni2003; Fadel et al., Reference Fadel, Bettendorff, Herrmann, de Azevedo, Oliveira, Yamane and Wüthrich2005). In addition, the presence of two histidine residues could provide an extra positive charge to these molecules in physiological pH and, especially, in the pH of the medium (Marinovic et al., Reference Marinovic, Dal Mas, Monte, Felix, Campeiro, Hayashi, Gopalakrishnakone, Inagaki, Mukherjee, Rahmy and Vogel2016).
In the present study, the ability of a new synthetic disulphide-less crotamine, i.e. the linear version free of the three S–S bonds, was evaluated for its ability to form a complex with DNA molecules. It is known that crotamine binding to nucleic acids seems to rely on electrostatic interaction; nucleic acids have a net negative charge while crotamine possesses an overall positive surface. One region, in particular, was suggested due to being potentially involved in the interaction with DNA. The region is comprised of the sequence RWRWK (Arg–Trp–Arg–Trp–Lys) and corresponds to residues 31–35 of NCr (Chen et al., Reference Chen, Hayashi, Oliveira and Karpel2012). In the linear disulphide-less peptide, this sequence was not altered, therefore preserving the ability of dLCr to bind to DNA. The rational to use disulphide-less crotamine as the delivery tool for embryo transfection instead of the native disulphide-bond folded crotamine relied on the fact that CPPs should make a complexation between the peptide and DNA complex that was strong enough to permit stable uptake through the zona pellucida and/or cell membrane translocation, but should be sufficiently weak to permit the release of DNA though dissociation into the cell cytoplasm to enable it to traffic to the nucleus. In our previous studies (Campelo et al., Reference Campelo, Pereira, Alcântara-Neto, Canel, Souza-Fabjan, Teixeira, Camargo, Melo, Rádis-Baptista, Salamone and Freitas2016a, Reference Campelo, Canel, Bevacqua, Melo, Rádis-Baptista, Freitas and Salamone2016b), bovine embryos were used as a model for gene transfer with DNA–NCr complexes. However, the use of the DNA–NCr complex did not improve the transgene expression rate, supposedly because of strong DNA–peptide binding within embryo cells (Campelo et al., Reference Campelo, Canel, Bevacqua, Melo, Rádis-Baptista, Freitas and Salamone2016b). Therefore, it was desirable to evaluate whether modified crotamine peptides, such as dLCr could reduce the binding strength of DNA–peptide and circumvent such physicochemical behaviour.
In fact, it was observed here that the capacity of complexation with DNA of dLCr was reduced when compared with NCr. In experiment 1, stoichiometry of DNA–dLCr and DNA–NCr complexes at the same proportion (1:10) was analyzed and complexed DNA was significantly reduced (34.9% vs 68.1%). The DNA–dLCr complex achieved similar complexation for 1:10 DNA–NCr (81.0% vs 68.1%) only at the 1:25 proportion, i.e. using approximately 2.5 times higher dLCr than NCr peptide mass for the same mass of plasmid DNA. It was expected that changes in the primary sequence, by substitution of cysteine residues in crotamine with 2-aminobutyric acid residues (Abu), would not alter membrane translocation, but facilitate peptide:DNA dissociation and preserve the accumulation patterns in cell cytoplasm.
Nevertheless, linear peptides usually have lower stability due to higher liability to proteases when compared with more compact disulphide-bond-stabilized structures, as for native crotamine (Marinovic et al., Reference Marinovic, Dal Mas, Monte, Felix, Campeiro, Hayashi, Gopalakrishnakone, Inagaki, Mukherjee, Rahmy and Vogel2016). Despite this concern, it was verified that the DNA–dLCr complex was highly stable in embryo culture medium and any degradation in peptide–DNA was observed even after incubation for 6 h (61.3% at 360 min vs 60.5% at 0 min). The stability of a given CPP in complete culture medium and in the presence of serum is a highly useful property in gene delivery (Rádis-Baptista et al., Reference Rádis-Baptista, Campelo, Morlighem, Melo and Freitas2017). Here, the potential toxic effect of dLCr on IVF bovine embryos was also evaluated and a significantly higher concentration of dLCr (12.5 µM) did not cause any deleterious effect upon IVF embryo development. Even at higher concentrations of dLCr (25 and 50 µM), toxicity in post-IVF early bovine embryos was undetectable. NCr has been extensively tested on eukaryotic cells such as mouse embryonic stem cells, human carcinoma HCT116 cells and CHO-K1 cells (Rádis-Baptista and Kerkis, Reference Rádis-Baptista and Kerkis2011). Specifically, mammalian embryos were also used as model to detect possible toxic effects of crotamine. However, for murine and bovine species (Kerkis et al., Reference Kerkis, Kerkis, Rádis-Baptista, Oliveira, Vianna-Morgante, Pereira and Yamane2004; Campelo et al., Reference Campelo, Pereira, Alcântara-Neto, Canel, Souza-Fabjan, Teixeira, Camargo, Melo, Rádis-Baptista, Salamone and Freitas2016a) no embryotoxicity was noticed when using native crotamine ranging from 1 µM to 10 µM, for up to 24 h. Overall, in agreement with these data and reports, native and linear crotamine are harmless to embryo cells in micromolar concentrations (< 50 µM for bovine and < 10 µM for mice).
Cytoplasmic microinjection of plasmids in zygotes is a simple alternative to drive ectopic expression of exogenous DNA in embryos (Iqbal et al., Reference Iqbal, Barg-Kues, Brol, Bode, Niemann and Kues2009). The development of a simple and effective method that could allow the delivery of exogenous DNA into the zygote’s cell membrane, through the zona pellucida, would be a great advantage to transgenic technologies, especially to the recent introduced engineered site-directed nucleases (ESDNs) methodologies, such as the CRISPR/Cas9 system (Kim, Reference Kim2016). These new technologies may require the injection of high concentrations of the transgene construct. In this scenario, improvements, such as using DNA construction in association with substances that enable further increase in efficiency of cytoplasmic microinjection, are a great advantage. For example, injection of DNA–liposome lipoplexes into IVF zygotes increased the rate of GFP+ blastocysts to 12% in cattle (Vichera et al., Reference Vichera, Moro and Salamone2011). In accordance, here it was evaluated if enhancement in efficiency of cytoplasmic microinjection of DNA carrying a GFP reporter could be achieved by associating exogenous DNA with dLCr by a non-covalent interaction. In vitro development parameters and fluorescence for GFP of IVF bovine embryos was compared with microinjection of DNA alone and IVF control groups.
Cleavage and blastocyst production at D7 and D8 were lower in the microinjected group than in the IVF control group, demonstrating, as expected, a possible negative effect of microinjection upon embryo development. Nevertheless, embryo development parameters were similar between embryos microinjected with DNA–dLCr or with DNA alone. Therefore, no detrimental effect was detected by using dLCr complexed with DNA for microinjection of post-IVF bovine embryos. A study using the same GFP plasmid construct with a microinjection semi-automated system was carried out to determine the most appropriate microinjection time, concentration and volume in IVF and parthenogenetic pig embryos. In this study, microinjection also reduced the developmental competence of injected zygotes, even when using the best conditions defined by the study (Malaweera et al., Reference Malaweera, Ramachandra, Wu, Oh, Kim, Kim, Jang and Cho2014). In contrast, a study in buffalo using an ApaL I linearized pEGFP-N1 plasmid, but with a manual microinjection system, resulted in cleavage and blastocyst development rates similar to those of non-injected IVF embryos (Meng et al., Reference Meng, Li, Wang, Qin, Oback and Shi2015).
Concerning the expression of GFP, a low rate was achieved (4.6% at D5 and 2.8% at D7) for group of embryos microinjected with plasmid DNA alone. This reduced rate could be explained by the low chance and the efficiency by which embryos normally express an exogenous cassette. In a correlated work, no GFP-expressing IVF bovine embryos were obtained after sperm-mediated gene transfer treatment, when exogenous DNA quantification could be assessed by real-time PCR (Campos et al., Reference Campos, de Leon, Komninou, Dellagostin, Deschamps, Seixas and Collares2011). As in our study, this group used a pEGFP-N1 plasmid containing the CMV promoter. Previously, the CMV promoter was thought to present low efficiency (Rieth et al., Reference Rieth, Pothier and Sirard2000) and, in this case, an additional CMV enhancer would be necessary to improve GFP expression. Moreover, injected DNA should be able to traffic to the nucleus and to be expressed even if DNA is integrated into the genome, randomly, and in a non-active transcription region of the chromosome (Hacker and Balasubramanian, Reference Hacker and Balasubramanian2016). This event is a significant drawback for genetic engineering regarding stable insertion of exogenous DNA in the genomes of farm animals, and an essential step for production of transgenic animal (Garrels et al., Reference Garrels, Ivics and Kues2012) that could be better achieved by CRISPR/Cas 9 technology.
When comparing embryos microinjected with plasmid alone or with the DNA–dLCr complex, the rate of DNA expression of exogenous DNA in bovine embryos was not comparatively improved. The high affinity for DNA and binding capacity of dLCr might again restrain the release of the GFP plasmid gene in the complex for protein expression, similar to found previously when using native crotamine to induce exogenous DNA expression in bovine embryos (Campelo et al., Reference Campelo, Canel, Bevacqua, Melo, Rádis-Baptista, Freitas and Salamone2016b). Therefore, it was hypothesized that dLCr remains complexed to exogenous DNA even inside the cytoplasm of embryo cells, and this possibly led to very low GFP expression and detectable fluorescence.
The results indicated that dLCr effectively formed a complex with DNA in a concentration- and time-dependent manner. Its application in embryo culture medium is possible for concentrations up to 50 µM for 6 h. However, transgene expression was not comparatively improved using dLCr complexed with a GFP reporter plasmid DNA. In conclusion, dLCr formed a stable complex with DNA, in the presence of complete embryo medium and was not embryotoxic. Substitution of the reporter gene will be under consideration for further utilization of dLCr as a tool to carry genes intracellularly and to improve heterologous gene expression. Additionally, the dLCr peptide sequence should be redesigned to improve GFP expression.
Acknowledgements
The authors are thankful to Dr Eduardo B. Oliveira (University of São Paulo, Ribeirão Preto, Brazil) for supplying native crotamine.
Financial support
This work was supported by the following Brazilian funding agencies: the Cearense Foundation for Scientific and Technological Development Support (FUNCAP, Pronex Programme), the National Council for Scientific and Technological Development (CNPq) and the Coordination for the Improvement of Higher Level or Education-Personnel (CAPES, Toxinology Programme).
Conflicts of interest
The authors have no conflicts of interest to declare.
Ethical standards
All animal experiments in this study were approved by the Ethics Committee on Animal Use of State University of Ceará (1672976/2017).
Supporting information
Supporting information of an MP4 video file is available for this paper.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0967199419000716
