|Home | About | Journals | Submit | Contact Us | Français|
Sperm-mediated gene transfer (SMGT) is a simple and efficient method for producing multitransgenic organisms. Until now, the exogenous DNA uptake efficiencies have been quantified, performing coincubation of spermatozoa with 3H-DNA. This method has significant limitations; from a researcher's point of view, radioactivity-based experiments are hazardous and require specialistic skills, and in technical analysis, the signal does not allow the simultaneous discrimination of two or more types of labeled constructs. Considering these remarkable points, the present work aims to develop a method for differential uptake quantification of various transgenes alternative to the use radioactive material. The main approach relies on fluorescent-specific peaks for each construct, and their diminution during the sperm—DNA-coincubation phase. The obtained results were confirmed by real-time PCR analysis and fluorescence microscopy imaging. This method becomes of primary importance when the SMGT technique has to be applied on various constructs, as it allows preliminary conclusions to be drawn about multiple transgenesis events and to approach further research about eventual sperm membrane preferences in sequences or structures for constructs.
Sperm-mediated gene transfer (SMGT) for creation of transgenic animals is an emerging technique, as documented by the increasing number of scientific publications about this topic in many species.1,2 In past decades, we optimized the technique in large animal species, i.e., bovine and swine,3 and then we obtained many successful productions of transgenic pigs for the human inhibitor of complement cascade (human decay accelerating factor).4,5
Until now, the exogenous DNA uptake efficiencies have been quantified, performing coincubation of spermatozoa with 3H-DNA and evaluating the resulting cpm in a β-counter before and during coincubation. The literature shows how the method of uptake quantification using DNA labeled with tritium proved to be absolutely reliable and effective.6,7 This, however, has a limitation inherent to the technology itself; the signal does not allow the simultaneous discrimination of two or more types of labeled constructs. Moreover, radioactivity is well known to be biologically hazardous, so it is desirable to replace it whenever possible.
Given the feasibility demonstrated in our previous studies, with the creation of multitransgenic pigs “one-step” through SMGT,8 the need to ascertain the distribution of the different constructs in the sperm population becomes of prime importance. Such a method should facilitate the monitoring of the uptake of multiple constructs during SMGT spermatozoa—exogenous DNA-coincubation phase—and at the same time, it should not cause significant side-effects on sperm. Experimental procedures should maintain spermatozoa close to SMGT operating conditions to gain reliable data. Considering these requirements, the present work aims to develop and validate a method for fluorescent uptake quantification of various transgenes by pig spermatozoa in real time.
Plasmids pT7T3-18U (GE Healthcare, Pittsburgh, PA, USA; 2.8 kb long), pGEM-9Zf(−) (Promega, Madison, WI, USA; 2.9 kb long), and pUC18 (Fermentas, Vilnius, Lithuania; 2.7 kb long) were used in sperm/circular DNA uptake analysis.
Fluorescent labels, specific for each construct, were added through a nick translation reaction. The reaction protocol of the nick translation kit (Amersham Nick Translation Kit N5000, GE Healthcare) had some adjustments to maximize the efficiency of the labeling reaction. Briefly, 1 μg DNA was diluted to a concentration of 200 ng/μl in molecular-grade water. DNA was placed in a clean microcentrifuge tube, and the reaction mix was added as follows: 7 μl dNTPs, dCTP excluded (dATP, dTTP, dGTP, 300 μM each); 1 μl fluorescent-labeled dCTP (10 mM); 10 μl enzyme mix; and water to a final volume of 50 μl. Fluorescent-labeled nucleotides used were: Cy3-dCTP (Cy3-AP3-dCTP, Amersham, Pittsburgh, PA, USA) for pT7T3-18U, max absorption at λ = 550 nm; Cy3.5-dCTP (Cy3.5-AP3-dCTP, Amersham) for pGEM-9Zf(−), max absorption at λ = 581 nm; Cy5-dCTP (Cy5-AP3-dCTP, Amersham) for pUC18, max absorption at λ = 649 nm. The reaction proceeded at 15°C overnight. According to the protocol, the reaction was stopped by the addition of 5 μl EDTA (0.2 M, pH 8.0).
To remove unincorporated fluorescent nucleotides and to concentrate labeled DNA, ethanol (EtOH) precipitation was done. A 50-μl vol 4 M ammonium acetate was added to the reaction volume. Then, 4 vol (200 μl) 90% pure EtOH was added to the reaction tube. The solution was chilled and rested at −20°C for 2 h. The reaction tube was then centrifuged at 14,000 g for 20 min at 4°C. The supernatant was discarded, and the pellet was washed with 5 vol (250 μl) ice-cold 70% EtOH. Following another 5-min centrifuge (14,000 g at 4°C), the supernatant was discarded again. Reaction tubes were air-dried, resuspended with 3.5 μl swine fertilization medium (SFM), and stored at −20°C.9
Two adult boars (Sus scrofa, Large White) of proven fertility were used as spermatozoa donors. The same boars were used previously in our studies based on 3H-DNA uptake.10 Parameters relevant to sperm quality were monitored during standard SMGT protocol on three occasions, in particular, after the first dilution (D) and immediately before (T0) and after 60 min of coincubation with DNA (T60). Fluorescent DNA quantification was performed at each experimental point (T0, T5, T15, T30, T45, T60).
The SMGT procedure was performed as described previously.8,9 Briefly, a sperm-rich fraction was collected from the selected donor, and seminal fluid was removed by carefully washing spermatozoa in SFM prewarmed to 37°C [SFM: 11.25 g glucose, 10 g sodium citrate (2H2O), 4.7 g EDTA (2H2O), 3.25 g citric acid (H2O), 6.5 g Trizma, adjusted to pH 6.8] following collection. Spermatozoa in SFM were diluted 1:20, and sperm quality analyses were performed (D). Semen was centrifuged (800 g, 10 min, 25°C), and the pellet was resuspended with SFM and centrifuged again. Washed spermatozoa were counted in a Thoma chamber and finally, diluted (SFM) to 5 × 108 sperms/ml (T0).
Spermatozoa viability was assessed incubating 50 μl sperm samples (D, T0, and T60) with 4 μl propidium iodide stock solution and 4 μl SYBR-14 stock solution, both obtained from the Live/Dead sperm viability kit (Molecular Probes, Eugene OR, USA), for 5 min in the dark. A 10-μl drop was placed on a slide, coverslipped, and then examined with a fluorescence microscope (Eclipse E600, Nikon, Chiyoda-Ku, Tokyo, Japan). At least 200 spermatozoa were counted for each slide.
Sperm motility of each sample was assessed subjectively by the same trained operator at 37°C (Raw Motility and Viability, assessed as described previously10) on a phase contrast microscope (Diaplan, Leitz, Germany).
SMGT uptake conditions were optimized for the fluorescence method: 5 × 106 sperms/1 μg DNA for each labeled construct/60 μl SFM. DNA and SFM were placed in a 50-μl cuvette (UVette, Eppendorf AG, Hamburg, Germany), and a read-out was taken before (T0) and immediately after adding spermatozoa (T5).
The whole cuvette was centrifuged for 1 min at 900 g through a cuvette-designed adapter, and as spermatozoa were still at the cuvette bottom, three spectrophotometric (GeneQuant 1300, GE Healthcare) read-outs of labeled DNA in suspension were taken (T5). Spermatozoa were then gently resuspended. For each experimental point (T15, T30, T45, T60), the same operations were repeated.
At the end of labeled DNA—sperm coincubation (T60)—a 10-μl drop was placed on a slide, cover-slipped, and then examined with a fluorescence microscope (Eclipse E600, Nikon) equipped with a digital camera. A rhodamine filter allowed for the investigation of cyanine-labeled DNA in the sperm cell.
To perform a qualitative and semiquantitative analysis of DNA uptake by spermatozoa, total DNA were extracted from the sperm after 5 min of coincubation (T5) and at the end of coincubation (T60). Each 50 μl sample (5×106 sperms) was washed with 1 ml SFM and centrifuged 4 min at maximum speed, and supernatant was discarded. DNA extraction was done with the extraction reaction kit (DNeasy Blood & Tissue Kit, Qiagen, Valencia, CA, USA), according to the manufacturer-specific protocol (purification of total DNA from animal sperm using the DNeasy Blood & Tissue Kit; protocol 2). Extracted DNA was quantified and used for real-time PCR. Several reactions have been set up for quantitative analyses. All of them made use of iCycler program suite (BioRad, Hercules, CA, USA) and the primer designer software Beacon Designer 3 (BioRad; Table 1). In every reaction, a genomic control on the HPRT gene was also considered. Real-time PCR was performed in the iCycler thermal cycler (BioRad) using SYBR Green I detection. A master mix of the following reaction components was prepared to indicate end-concentrations: forward primer (0.2 μM), reverse primer (0.2 μM), IQ SYBR Green BioRad Supermix (BioRad), and sample DNA (100 pg). All of the samples were analyzed in duplicate.
The real-time PCR protocol used was the following: initial denaturation for 3 min at 95°C, 40 cycles of 95°C for 15 s, and 60°C for 30 s, followed by a melting step with slow heating from 55°C to 95°C with a rate of 0.5°C/s.
The amount of DNA uptake for each construct was calculated through the subtraction from the supernatant fraction value of each Cy at T0. Background was obtained from the mean of λ = 520 nm read-outs. Data showing excessive background values, significantly different from the mean, were rejected. Single fluorophore absorbance peak at T0 was considered as 100% of DNA in suspension; all of the percentages of uptake are calculated through the proportion from T0 and the read-outs at specific time-points. Three trials, in different times, were performed on the semen of each donor.
Data about sperm viability and motility are reported in Figure 1. The fluorescent DNA uptake quantification method did not negatively affect sperm motility. Also, viability had no significant decrease from experimental points D to T60.
At the end of DNA—sperm coincubation—spermatozoa were observed for fluorescence to check for the presence of labeled plasmids. More than 95% of observed spermatozoa showed cyanine fluorescent signals (Fig. 2).
The three exogenous constructs present were calculated as Δcomparative threshold (Ct) = (HPRT Ct−pXX Ct). As the HPRT, Ct were similar among samples; increased ΔCt values represent an increase in exogenous DNA uptake. The levels of pGEM-9Zf, pT7T3, and pUC18 DNAs recovered after spermatozoa extraction are shown in Figure 3. The relative amount of plasmidic DNA increased significantly at the end of the coincubation phase (T60). There were no differences among the three plasmid reactions.
Experimental conditions allowed for the simultaneous read-outs for each labeled construct at every time-point, showing a progressive diminution in cyanine absorption peaks. Collected data allowed for the calculation of DNA uptake for each cyanine-labeled construct in all experimental time-points. A representative uptake percentage plot is represented in Figure 4. Every construct was taken in from the solution by >70 ± 8%.
This research succeeded in developing an effective method for the concurrent quantification of multiple DNAs uptake during SMGT and can be considered as the first alternative to radioactivity-based analysis. Sperm quality analyses showed how the application of the fluorescent-uptake method did not harm spermatozoa; in fact, viabilities were very close among them and to the ones observed in standard application of SMGT protocols. Furthermore, the centrifuge settings were optimized to achieve a fast displacement of the sperm to the bottom of the cuvette and at the same time, to preserve spermatozoa integrity.
The fluorescent labels have been chosen because of their high sensitivity, low nonspecific binding, pH insensitivity, and spectral resolution. This last feature is the most important, as it allows the identification of three distinct signals in a single spectrophotometric lecture. To obtain an absorbance peak above the device limit of detection, it was necessary to work with 1 μg of each label. The final amount of DNA is approximately tenfold greater than that described previously.5 This is not a significant hurdle, as we demonstrated recently that even with a 20-fold larger amount of DNA, sperm quality parameters were not affected significantly.10
Besides reliability, the key to this method is centrifugation and coincubation directly inside the cuvette; this way, suspension splitting is avoided, and no bias is introduced during data analysis. In addition, the entire method relies on a simple principle: before the addition of spermatozoa, all of the labeled DNA molecules are in solution, and after the beginning of coincubation, the centrifugation step allows detection of a progressive decrease of the absorption peaks as a result of DNA-selective binding to spermatozoa, displaced at the bottom of the cuvette by centrifugation. The technique has no inner limits, as many different constructs are detectable as fluorophores available. Although we observed in all of the performed experiments that in small time lapses, absorbance kinetics might be quite different (data not shown), the final DNA uptake for each construct was always >70 ± 8%.
Uptake data were confirmed through the observation of fluorescent signals on the sperm surface, which resembled the colocalization of exogenous DNA on the sperm head described previously and through real-time PCR analysis. PCR analysis showed that the presence of the three plasmids is great and reflected fluorescence data in both experimental points.
The importance of the present work is straightforward. Smith and Spadafora11 have previously described the broad use of DNA incubation as the most common methodology to induce seminal plasma-free sperm to internalize transgenes. The scientific literature has more than 30 reports of successful in vitro uptake of DNA by sperm cells in various species,12 from sea urchin to honey bee, passing through various mammals, including several farm animal species. The method developed in this research becomes of crucial importance, as it is not directed uniquely to swine sperm, but rather, its use can include many other species.
Moreover, considering the creation of multitransgenic pigs one-step through SMGT, the need to ascertain the relative distribution of the different constructs in the population of sperm is of prime importance. In addition, many scientific publications pertaining to SMGT-treated semen had to resort to split transgene analysis,12,13 and applying the present, new method, it is possible to investigate all of the transgene behaviors at the same time. This method constitutes a powerful tool for further studies, such as monitoring eventual sperm membrane preferences in sequences or structures for different constructs at the same time. This method of differential quantification seems to be perfectly compatible with SMGT and at least allows the replacement of radioactivity, saving time and protecting laboratory personnel during analysis.
Professor Marialuisa Lavitrano provided the financial support through the European Commission's Sixth Framework Programme “Xenome” (contract no. LSHB-CT-2006-037377); grants were from the Research Funding Office 60% and by Consorzio Interuniversitario per i Trapianti d'Organo. We thank Dr. A. Zaniboni for his precious contribution.