Electroporation of bovine spermatozoa to carry foreign DNA in oocytes

In the present study, electroporation was used to test the ability of spermatozoa to carry foreign DNA into the bovine oocytes. Frozen-thawed bovine spermatozoa (10(7)/ml) were electroporated using six different combinations of voltage (500, 1,000, or 1,500 V) and capacitance (1 or 25 microFarads) in the presence of 1 mg/ml of plasmid pRGH527. The portions of plasmids retained by sperm cells after three washings (stable for ten washings) were 4.3, 5.5, 5.1, 6.0, 6.8, and 5.8% for 1 microFarad, 500, 1,000, and 1,500 V and 25 microFarads, 500, 1,000, and 1,500 V, respectively. Nonelectroporated cells have retained only 1% of plasmids. In the same experiment, electroporated spermatozoa were acrosome reacted by treatment with ionophore A23187 to evaluate the fraction of marked plasmids joined at the acrosomal membrane. The results show that 3.5, 5.0, 4.4, 5.0, 6.3, and 4.4% remain tied to the ionophore-treated sperm. Only 0.7% of plasmid was retained after removal of the acrosome of nonelectroporated cells. Acrosome reaction was not significantly induced by the electrical field (EF) (P less than 0.005). EF decrease motility significantly for greater than 100 V in 0.3 M mannitol (M) and mannitol-TALP (MT) (1/1) media and greater than or equal to 500 V (P less than 0.05) in TALP medium. The retained plasmid rate was compared between TALP medium M and MT media and resulted in a percentage of 1.0, 2.5, 6.5 at 1 microFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 microFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 microFarad, 500 or 1,000 V, 25 microFarad, 500 V or 1,000 in TALP medium hold plasmids in proportion of 5.2, 5.4, 7.4, and 6.0%. Electroporation above 100 V in M and MT killed the cells. In a part of this experiment, spermatozoa electroporated in the presence of radiolabeled plasmids have been treated with DNase I and results revealed that 35, 28, 54, 58, and 3% of marked DNA remains in sperm cells following digestion after electroporation in TALP (1,000 V, 1 and 25 microFarads), M medium (100 V, 1 and 25 microFarads), and control, respectively. Using in vitro matured bovine oocytes, the electroporation conditions were correlated with the fertilization rate (85% for control and 55% for electroporated spermatozoa). Autoradiography of embryos following fertilization indicated the presence of plasmids in the cytoplasm and in the zona pellucida.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events

There are several methods of modifying bovine genomes. Pronuclear microinjection is more widely used but it is still to be improved. Searches for alternatives have lead to the development of new methods including SMGT (Sperm Mediated Gene Transfer), in which live spermatozoa are used as vehicles for DNA delivery during in vitro fertilization. In previous studies, we presented evidence that a highly repetitive Alu-like repeat favours transgenesis by homologous recombination (HR). Up to 60% integration via HR was obtained following pronuclear microinjection of a Pst1 beta-actin GFP DNA construction. In the present study, we show that HR-mediated integration is also possible using SMGT, since bovine spermatozoa electroporated with the same DNA construct are able to transfer it to a high proportion of embryos obtained by in vitro fertilization. Swim-up selected bovine spermatozoa were mixed with the Pst1 beta-actin GFP construct (6 x 10(6) spermatozoids were incubated with 600 ng of muDNA), submitted or not to electroporation (300 V, 25 F) and treated or not with DNase I. The process of electroporation itself did not affect in vitro embryonic development. However, oocytes fertilized with electroporated DNA-treated spermatozoa developed beyond the 16-cell stage in proportions that were significantly lower (27% with Pst1 beta-actin GFP and 34% with beta-actin GFP) compared to the control without DNA (44%). On the other side, the use of electroporation significantly increased the uptake of DNA. The number of homologous recombination events detected by PCR went from 3.5% without electroporation to 46.5% after electroporation. In conclusion, our results confirm that spermatozoa electroporation combined with homologous recombination in a highly repetitive Pst1 sequence is a feasible method to obtain transgenic bovine embryos.     

电脉冲作用将外源基因导入稀有(鱼句)鲫精子的研究

将稀有鲫 (Gobiocyprisrarus)精子与重组质粒pCAhLFc线性DNA混合温育 ,经电脉冲处理后与卵子受精 ,孵化出苗。从鱼苗中提取DNA ,经PCR检测 ,2 5 .5 %～ 6 6 .7%鱼苗带有外源基因。在显微镜下观察经电脉冲处理过的精子 ,发现其活力有不同程度下降 ,受精率也有不同程度下降 ,说明不同的电脉冲条件对精子有不同程度的损害作用。精子与外源DNA混合温育 ,经电脉冲处理后 ,用DNA外切酶消化后 ,提取精子DNA ,经PCR检测 ,仍有阳性电泳带 ,证明电脉冲可以促使稀有鲫精子摄入外源基因     

Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta)

We investigated gene transfer in abalone via electroporated sperm. The mobility of sperm electroporated either in seawater or in marine invertebrate physiological solution was as good as that of the control group. The fertilization rate reached as high as 94.7--99.6% (93.0-- 99.7% for the control group) when 200 eggs were fertilized by 106 or 107 sperm treated with electroporation at 10 kV and 27 pulses for six cycles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 g and at voltages ranging from 2 to 10 kV, at 27 or 211 pulses for six or 12 cycles showed no differences from the control sperm. After DNase digestion, the genome of the electroporated sperm was analysed by polymerase chain reaction, and it was shown that a 138-bp product was amplified, corresponding to the transgene's amplification product. Southern blotting also showed that a positive band located at the same position as that of opAFP-2000CAT was found in the electroporated sperm after DNase treatment. Analysis by PCR of the genome isolated from a trochophore-stage abalone larva, derived from sperm electroporated with 3.2 g opAFP- 2000CAT, showed the existence of foreign DNA in 13 out of 20 examined samples (65%). The integration of the transferred DNA into the genome of transgenic abalone was also shown by Southern blot analysis. Furthermore, CAT activity was positive for the experimental larvae, but the level of CAT expression was lower than that of larvae derived from sperm electroporated with pCAT- Control vector, driven by SV40 promoter and enhancer sequences. These results demonstrate the potential for the use of sperm as mass gene transfer strategy in marine mollusks such as abalone     

Electroporation of cells

By measuring uptake of the membrane impermeable dye. phenosafranine, it can be shown that the plasma membrane of intact cells within cell aggregates can be reversibly permeabilized by electroporation. However, the plant cell wall is a barrier to DNA uptake by intact cells, although under certain circumstances expression of DNA, electroporated into intact cells, can be demonstrated. The level of expression is about 20–50 times lower than that obtained by electroporation of protoplasts, and depends on cell wall properties and pretreatments of cell aggregates. In contrast, efficient transformation of whole cells of bacteria and yeasts can be achieved by electroporation. Factors which influence DNA transfer into whole plant cells and the possibility of stable transformation are discussed.     

Optimization of Electrotransfection Conditions of Mammalian Cells with Different Biological Features

We introduced eukaryotic expression plasmid pEGFP-N1 encoding green fluorescent protein (GFP) genes into cells with different biological features through electroporation. The effects of conditions, including voltage, capacitor flow, pulse cycle, DNA dosage and buffer, on transfection efficiency were investigated based on fluorescent microscopy and posttransfection survival rate of cells by staining with trypan blue. Better electrotransfection outcomes were achieved in the following epithelial cells: Vero cells at 300?V/850???F, PK15 cells at 300?V/500???F, MDCK cells at 200?V/600???F, F81 cells at 200?V/500???F, cancer cells MB49 at 300?V/400???F, Hela cells at 200?V/450???F, HF-29 cells at 300?V/800???F and B16F1 cells at 200?V/650???F. Among fibroblast cells, better electrotransfection was achieved in BHK21 cells at 300?V/600???F and ST cells at 200?V/750???F. RPMI-1640 medium without antibiotics and serum demonstrated higher electrotransfection efficiency and cell survival rate than other cell culture media as electroporation buffer. Our findings further prove that electroporation transfection is an effective method for genetic transfection. Cells with different biological features require varying transfection conditions to obtain higher transfection efficiency of target genes.     

Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii.

Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501. The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation. For example, Log, 4.44 transformants microgram-1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 microF did not produce any transformants. Cell survival was also poor at high voltages. A. vinelandii transformants were not recovered on N-free agar medium. In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 microF; 2500 V, 3 microF; and 1500 V, 25 microF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.     

Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

Previous studies have shown greater fluorophore uptake during electroporation on the anode-facing side of the cell than on the cathode-facing side. Based on these observations, we hypothesized that hyperpolarizing a cell before electroporation would decrease the requisite pulsed electric field intensity for electroporation outcomes, thereby yielding a higher probability of reversible electroporation at lower electric field strengths and a higher probability of irreversible electroporation (IRE) at higher electric field strengths. In this study, we tested this hypothesis by hyperpolarizing HL-60 cells using ionomycin before electroporation. These cells were then electroporated in a solution containing propidium iodide, a membrane integrity indicator. After 20 min, we added trypan blue to identify IRE cells. Our results showed that hyperpolarizing cells before electroporation alters the pulsed electric field intensity thresholds for reversible electroporation and IRE, allowing for greater control and selectivity of electroporation outcomes.     

Introducing foreign genes into fish eggs with electroporated sperm as a carrier.

A new method has been developed for introduction of foreign genes into fish eggs. The procedure is based on the incubation of fish sperm cells suspended in dilute citrate solution with plasmid DNA, followed by application of high-field-strength electrical pulses (electroporation) to increase DNA binding., uptake, or both. Tissue homogenates and genomic DNA extracts of free swimming fry developed from eggs fertilized with treated sperm was tested to evaluate the efficiency of gene transfer. Dot blot hybridization and gene expression assay demonstrated the presence and expression of the reporter genes introduced in 2.6 to 4.2% of several hundreds of tested larvae of common carp (Cyprinus carpio L.), African catfish (Clarias gariepinus), and tilapia (Oreochromis niloticus). No transgene has been found in the fry resulting from parallel experiments without sperm electroporation. This is the first report on successful application of electroporated sperm cells for production of transgenic fish.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.     

Influence of Polyethylene Glycol on the Size of Schizosaccharomyces pombe Electropores

The role of polyethylene glycol (PEG) in the transformation of Schizosaccharomyces pombe by electroporation is investigated by fluorescein isothiocyanate-dextran uptake and transformation studies. It is shown that when S. pombe cells are electroporated in the presence of PEG, the permeability state created is sustained until removal of PEG. In addition, the permeability of electroporated S. pombe envelopes is further increased with longer incubation times in PEG. The increased permeability is apparently a result of enlarged pores (electropores) due to the presence of PEG. Comparison of a heat pulse transformation protocol with electroporation suggests a second role for PEG in the uptake of macromolecules. Since pores are not thought to be created during a heat pulse, the PEG may be facilitating the uptake of plasmid DNA. This facilitation of uptake would also be expected to affect DNA uptake by electroporated cells.     

Effect of microinjection and two types of electrical stimuli on bovine sperm-hamster egg penetration

These experiments were designed to test the effects of an electrofusion and an electroporation pulse on bovine sperm-hamster egg development. In experiment 1, single motile sperm were injected into the perivitelline space of each egg. A 4,500 V/cm, 30 microseconds fusion pulse (FP) was applied while sperm-egg membrane contact was maintained. It was observed that single motile sperm were rendered immotile immediately after FP application whereas nonpulsed single motile sperm remained motile for up to 36 h postinjection. In addition, both motile and sonicated spermatozoa were injected directly into the ooplasm prior to receiving an FP to determine whether the FP was detrimental to sperm viability. In experiment 2, to induce the acrosome reaction, an 1,150 V/cm electroporation pulse was applied to washed bovine sperm suspended in TALP medium containing 5 mM Ca2+. Treated and nontreated sperm were coincubated with zona-free hamster ova, and sperm-pentrating ability was measured. Results from experiment 1 indicate that FP failed to induce sperm-egg fusion (0/69). FP did not, however, inhibit decondensation or pronuclear formation of sperm injected into hamster egg ooplasm. Single motile sperm injected into the ooplasm resulted in development of both pulsed (19/28) and nonpulsed (21/28) groups. Sonicated tail-free sperm heads injected into the ooplasm resulted in no detectable difference between treated (18/30) and nontreated (19/30) groups. In experiment 2, treatment of sperm with electroporation pulse +5 mM Ca2+ increased zona-free hamster ova penetration scores over nontreated sperm within bulls (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of Saccharomyces cerevisiae by electroporation

A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.     

Transformation of Saccharomyces cerevisiae by electroporation.

A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.     

Electrotransfer of [32P]NAD allows labeling of ADP-ribosylated proteins in intact Chinese hamster ovary cells

CHO-K1D cells electroporated in buffers containing [32P]NAD incorporated the label in a voltage-dependent manner. Electroporation with 650 V/cm at 1460 microF in Ham's F12 medium supplemented with 10 mM Hepes, pH 7.1, resulted in a greater than 20-fold increase in [32P]NAD uptake, while decreasing relative cellular survival by only 6%. Exposure of cells to gamma irradiation (20 Gy) prior to electroporation increased the steady-state level of poly(ADP-ribosylated) nuclear proteins two- to four-fold over that of unirradiated control cells. These data indicate that electrotransfer of [32P]NAD is a simple and rapid means of labeling the cellular NAD pool and should prove useful in the analysis of the relationship between poly(ADP-ribosylation) of nuclear proteins and DNA repair.     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate     

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 × 10⁶ cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.     

Electroporation of extraneous proteins into CHO cells: Increased efficacy by utilizing centrifugal force and microsecond electrical pulses

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.     

Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen

Cryopreservation imposes irreversible damage to sperm membranes, such as swelling and disruption of plasma and acrosome membranes, changes in membrane fluidity, altered influx of calcium, and changes in enzyme activity. Morphological integrity of the sperm plasma membrane has been widely studied using different techniques, including exposure of spermatozoa to hypoosmotic solutions (provides information concerning the biochemical activity of the sperm tail membrane), supravital test using eosin stain (yields information regarding sperm head membrane integrity), and Trypan-blue Giemsa stain (TBG; reveals both sperm plasma membrane and acrosome integrity). The objective of this study was to combine these tests in order to provide information about the integrity of the whole sperm surface, as well as acrosome status, and determine if the results of these tests were associated with sperm in vitro fertilizing ability. Stepwise regression analyses yielded a model in which fertility (maintain variable) was expressed as a combination of the results of different spermatological parameters (independent variables). The results of a test combining supravital eosin staining of samples previously submitted to hypoosmotic swelling test (STHOS) accounted for the greatest proportion of variation in fertilization rates (78%). Inclusion of the results of dual staining with TBG increased the proportion of variation in fertility rate that could be accounted for to 82%. Therefore, sperm plasma membrane integrity and function, and acrosome integrity can be considered important variables for normal sperm function and STHOST and TBG could be used for the prognosis of the potential fertility of bovine semen samples used for IVF or AI.