Polyethylenimine promotes sperm-mediated transgene and oligonucleotide delivery in abalone Haliotis discus hannai.

ABSTRACT In this study, a pCMV2-EGFP plasmid (Invitrogene), in which the enhanced green fluorescent protein gene under the control of the CMV2 promoter, was used as a reporter gene to evaluate the efficiency of the gene transferring techniques into Pacific abalone Haliotis discus hannai. The GFP reporter gene was transferred into abalone eggs with four methods: microinjection method, sperm vector method, polyethylenimine (PEI) method and polyethylenimine-mediated sperm-vector (PEI-SV) method. The results indicate that the PEI-SV method is more efficient and convenient in gene delivery into abalone eggs compared with the microinjection method and the conventional sperm vector method. A significant proportion (13.9%) of the fertilized eggs developed normally into blastomeres and expressed green fluorescent protein at high level after gene transfer with the PEI-SV method, and 7.6% of them survived to the trochophore stage. PCR analysis suggests that the mechanism of PEI-SV gene transfer method was that PEI/DNA was taken up by spermatozoa and carried into eggs during fertilization. In addition, PEI-mediated transport of FAM-labeled 10 mer oligonucleotide into abalone sperm cells resulted in almost 100% bright fluorescent nuclei. The PEI-SV gene transferring method can be an effective tool to transfer gene into other animal species.

KEY WORDS: polyethylenimine, gene transfer, microinjection, sperm, abalone, Haliotis discus hannai

INTRODUCTION

Abalone is a valuable marine shellfish with great economic importance in marine aquaculture. Haliotis discus hannai is the major commercial abalone species in the northeast coastal area of China. Because abalone grow very slowly, it generally takes 3-4 y of farming from seeding to harvesting, which greatly limits the productivity of abalone aquaculture. Transfer of growth hormone gene is a possible way to breed fast-growing abalone. Research on transgenic abalone is far from successful because of many difficulties in gene transferring technologies. One of the most intractable obstacles is the lack of a suitable method to transfer foreign genes into fertilized abalone eggs.

The first successful transfer of a rat growth hormone gene, under a metallothionein promoter, into a mouse, resulted in a dramatic increase in growth. This was performed using a microinjection method (Palmiter et al. 1982, Palmiter et al. 1983). Since then several other techniques for gene transfer such as sperm vector, electroporation, high-velocity microprojectile bombardment, and sperm electroporation, have been developed. None of these methods are appropriate for transferring foreign genes into marine shellfish species. Because of the high mortality of fertilized eggs and larvae, millions of fertilized eggs must be transferred to ensure the production of transgenic shellfish. Microinjection is still a general method with acceptable efficiency in transferring gene into most animal species. However, it is almost impossible to produce transgenic shellfish with the microinjection method. Generally, micromanipulation cannot produce enough embryos due to the difficulties of skills, the length of time required, and high mortality of the injected eggs. The ease of artificial fertilization of shellfish enables the use of sperm electroporation, a powerful tool for increasing efficiency and number of transgenic embryos. Although high gene transfer efficiency has been reported (Powers et al. 1995), sperm electroporation, as well as microinjection; are difficult to control, demand high skill levels, and are time-consuming methods, which limit their application to shellfish.

To enhance the efficiency and overcome the difficulties in transgenic shellfish research, a new gene transfer method--polyethylenimine-mediated sperm-vector (PEI-SV) is proposed and evaluated by transferring a green fluorescent protein reporter gene into the Pacific abalone, Haliotis discus hannai.

MATERIALS AND METHODS

Abalone

Mature male and female parental Pacific abalone Haliotis discus hannai individuals were obtained from Rongcheng Xunshan Abalone Hatchery in Shandong Province. After the ovulation or sperm ejaculation of the parental abalone, eggs and sperm were mixed together for fertilization when needed. The density of the eggs and sperm was controlled at approximately 1:10,000 to gain a high fertilization rate. The fertilized eggs were washed with filtered seawater 20 min after fertilization to remove excessive sperm and prevent them from decaying.

The Reporter Gene

A pCMV2-EGFP plasmid (Invitrogene) in which the enhanced green fluorescent protein gene was under the control of the CMV2 promoter, was used as a reporter gene to evaluate the efficiency of the gene transfer techniques. E. coli. DH5[alpha] was transformed with the pCMV2-EGFP plasmid. The expression of the green fluorescent protein gene in E. coli. DH5[alpha] was visualized through a fluorescent microscope. The plasmid was then propagated and extracted. Fifty [micro]g of the circular pCMV2-EGFP plasmid was cut with restriction enzyme EcoR I, purified with a plasmid purification kit (SANGON) and dissolved in 500 [micro]L of D[W.sub.2] for use.

The PEI/DNA Complex

Forty [micro]g of the linear pCMV2-EGFP plasmid DNA was diluted into 2 ml of D[W.sub.2]. PEI vector was used at 1:1 PEI/DNA ratios (W/W). The desired amount of 25-kDa polyethylenimine (Sigma) was added to the plasmid solution, mixed, vortexed, spun down, and incubated at room temperature for 10 min before use.

Gene Transferring

The experiment was designed in 5 groups: control group, microinjection group, SV group, PEI group, and PEI-SV group. The control group consisted of about 50,000 fertilized eggs, without any treatment. The development of fertilized eggs was observed microscopically and expression of GFP was monitored with an Olympus fluorescent microscope. All experiments were done in duplicate. The fertilization and survival rates were calculated for each group at 1 h and 16 h after fertilization, when the fertilized eggs began the first cleavage and became trochophores respectively. The expression of the GFP was monitored with fluorescent microscopy through the developmental process of the embryos from 1 to 6 h alter fertilization.

Microinjection Group:

The reporter gene was transferred with the microinjection method. Two [micro]g of the linear pCMV2-EGFP plasmid was diluted with microinjection buffer (7 mM Tris, 0.15 mM EDTA, pH 7.0) to a final concentration of 2 [micro]g/mL. Fertilized eggs were taken from the control group and 500 eggs were injected with 0.02 [micro]L [P.sub.CMV2]-gfp solution for each (Fig. 1A). Microinjection was performed using a Nikon micromanipulation system.

[FIGURE 1 OMITTED]

SV Group:

About 10,000 eggs were transferred with the conventional sperm-vector method. Four [micro]g of the linear plasmid pCMV2EGFP was diluted to 2 [micro]g/ml with D[W.sub.2], and mixed with the sperm at 1:50 volume ratio. The sperm/DNA mixture was incubated at room temperature for 10 min and mixed with the eggs for fertilization.

PEI Group:

About 10,000 eggs, taken from the control group, were transferred with the reporter gene using PEI as vector. The DNA/PEI mixture with 4 [micro]g of DNA was mixed with fertilized eggs at a 1:50 volume ratio, 10 min after fertilization.

PEI-SV Group:

About 10,000 eggs were transferred with the PEI-mediated sperm-vector method. The DNA/PEI mixture with 4 [micro]g of the linear plasmid DNA was added to the sperm at 1:50 volume ratio and gently mixed with a pipette. The sperm/DNA/PEI mixture was mixed with the eggs for fertilization immediately, by which the DNA/PEI complex was transferred into the eggs.

PCR Analysis of the GFP Reporter Gene

Polymerase Chain Reaction (PCR) technology was used to testify whether PEI can carry DNA into abalone sperm cells or not. Three sperm/PEl/DNA mixture samples were washed with D[W.sub.2] and sperm was collected after centrifuging three times to remove DNA molecules attached to sperm surfaces. Then DNA was extracted with Genomic DNA extraction kit (Sangon) and tested with PCR technology using a pair of GFP gene specific primers: P1: 5'-CCAACACTTGTCACTACTTT-3', P2: 5'-GCTTTGATTCCATTCTTT-3'. DNA extracted from normal abalone sperm and sperm/DNA mixture was used as a negative control, and plasmid DNA was used as a positive control. The PCR reactions were performed in a total volume of 20 [micro]L, using 100 nM of each primer, 200 mM dNTPs, in 1X PCR Buffer supplemented with Mg[Cl.sub.2] at a final concentration of 1.5 mM. The amplification was carried out on a MJ PTC- 100 thermal controller as follows: a 2 min 2predenaturing at 94[degrees]C, followed by 30 cycles (30 s at 94[degrees]C, 1 min 30 s at 60[degrees]C, and 3 min at 72[degrees]C). The reaction was completed by a final extension step at 72[degrees]C for 5 rain, then electrophoreses on a 1.5% agarose gel for 30 min at 5V/cm and visualized with UV light after EB staining.

Transferring Fluorescent Labeled Oligonucleotides

Five [micro]g of FAM-labeled 10 bp random sequence oligonucleotides were diluted into 200 [micro]L of D[W.sub.2]. PEI vector and used at 1:1 PEI/DNA ratios (W/W). The desired amount of 25-kDa polyethylenimine (Sigma) was added to the plasmid solution, mixed, vortexed, spun down, and incubated at room temperature for 10 min. Then the FAM-labeled oligonucleotides and the PEI/oligonucleotides mixture was added to the sperm cells, mixed, vortexed, spun down, and incubated at room temperature for 60 min. The sperm was mixed with eggs for fertilization, and then the sperms and the eggs were observed through fluorescent microscopy.

RESULTS

Fertility, Embryo Survival, and Expression of GFP

The fertilization rate, survival rate and the GFP expression rate for each group is shown in table 1. In the control group, 97.4% of the fertilized eggs cleaved, and 83.6% of them survived to the trochophore stage. The fertilized eggs used in the microinjection group were taken from the control group, so the fertility rate was also 97.4%. However, none of the 500 injected fertilized eggs cleaved 1 h after fertilization. Fluorescent microscopy revealed that none of them expressed GFP at 5 h after fertilization.

In the SV group, 95.2% of the fertilized eggs cleaved and 82.3% of them survived to the trochophore, but none of them expressed GFP. In this study, the conventional sperm vector method did not decrease the fertilization rate and the survival rate, yet it did not deliver GFP gene into the fertilized eggs successfully either. In PEI group, the fertilized eggs were also taken from the control group, so the fertility rate should also be 97.4%, whereas the survival rate to trochophore was only 52.7%, which was significantly lower than that of the control group. Fluorescent microscopy revealed 3.8% of them expressed GFP and a small proportion (less than 5%) of them expressed GFP in mosaic manner (Fig. 1B).

In the PEI-SV group, only 16.3% of the fertilized eggs cleaved by 1 h after fertilization, and 7.6% of them survived to trochophores by 16 h after fertilization. The fertilization rate and survival rate was significantly lower than the control group. The gene delivery efficiency was, however, significantly higher than the other groups: 13.9% of the fertilized eggs developed into blastomeres and expressed GFP at high level, at 5 h after fertilization (Fig. 1C). Compared with the PEI group and the SV group, it is obvious that PEI promotes the sperm-mediated gene delivery into the abalone eggs.

PCR Analysis of the GFP Reporter Gene

PCR technology was used to test whether PE1 could carry DNA into sperm ceils or whether it was attached to the cell membrane. The sperm/PEI/DNA mixture was washed three times with clean seawater and the sperm DNA was extracted and amplified with GFP gene specific primers. For all 3 samples tested, the strong PCR bands showed that the PEI/DNA complex did carry DNA into the sperm cell, or the complex attached to the cell membrane that cannot be washed off with water. The negative controls amplified with DNA extracted from normal sperm cells or sperm cell mixed with DNA without PEI treatment, showed no bands, indicating that the sperm cells did not absorb DNA by itself or DNA was degraded by nuclease without the protection of the PEI/DNA complex.

PEI Carried Oligonucleotides to Sperm Cells

Generally short oligonucleotides do not require a carrier to enter a cell through fluid-phase endocytosis. However lysosomal degradation remains a problem. Polyethylenimine has been shown to help oligonucleotides to reach their nuclear target (Boussif et al. 1995). We therefore tried PEI for carrying oligonucleotide into abalone eggs. In the control experiments, abalone sperm cells were incubated with FAM-labeled 10 met oligonucleotide (10 [micro]M) for 2 h, then rinsed and fixed. Fluorescent microscopy did not reveal any remaining cell-associated oligonucleotide or nucleoside, which may be a consequence of nuclease degradation. In sharp contrast, PEI-mediated transport of the same concentration of FAM-labeled 10 mer oligonucleotide into abalone sperm cells, resulted in almost 100% bright fluorescent nuclei (Fig. 1E). Using these sperm for fertilization, fluorescent microscopy revealed that the sperm with fluorescence attached to the membrane of the fertilized eggs (Fig. 1F), indicating that the sperm are still capable of insemination after PEI/DNA complex treatment.

DISCUSSION

In animals, microinjection was the earliest technique developed to introduce foreign DNA into fertilized eggs. However, it is a difficult and time-consuming technique that requires sophisticated skills. Because of the opaqueness, stickiness, and buoyancy of the embryos, the invisibility of the pronuclei, the toughness of the chorion, and the high mortality of injected eggs, this is not a suitable method for gene transfer in most shellfish species. Gene transfer into abalone fertilized eggs with microinjection technology is extremely difficult because the eggs are surrounded with a tough chorion membrane. If the chorion membrane is removed, the embryos become fragile and are easily destroyed by micromanipulation. The eggs are opaque and the pronucleus is invisible, which makes it almost impossible to inject the DNA solution into the male or female pronucleus of the fertilized eggs, which is generally required in gene transfer with microinjection. The yolk in the eggs easily flows out when the eggs are pierced with the glass micro pinhead (Fig. 1A), which could cause the high mortality of the injected eggs. These difficulties also limit the number of eggs that can be injected, even though millions of eggs can be produced by one single female abalone. In this research, the high mortality of the injected eggs and the lack of expression of GFP, indicate that microinjection is not suitable for transferring genes into abalone.

These problems are partially solved by the use of the electroporation method or the sperm electroporation method. High gene transfer efficiency was reported when a recombinant plasmid, containing a beta-galactosidase cassette, was introduced into fertilized eggs of the red abalone Haliotis rufescens by electroporation (Powers et al. 1995). The conventional sperm vector method is neither effective nor stable, but it is quite successful when combined with the electroporation method. Sperm electroporation is again a difficult-to-control, and skill-demanding method and its application to marine shellfish is limited because of the high electrical conductivity of seawater. To enhance the efficiency and overcome the difficulties in transgenic shellfish research, here we propose the polyethylenimine-mediated sperm-vector (PEI-SV) method. The results indicate that PEI can promote DNA transfer into abalone eggs, especially when combined with the sperm vector method. A significant proportion (13.9%) of the fertilized eggs developed normally into blastomeres and expressed green fluorescent protein at high level, 7.6% of them survived to the trochophore stage after gene transfer with the PEI-SV method.

PEI is an aqueous organic macromolecule with high cationic charge density potential, which can be complexed with DNA. PEI/ DNA complexes can be used for in vitro and in vivo gene delivery approaches. The excess of positive surface charges enhances the association of the complex with the plasma membrane of cells and facilitates their uptake by endocytosis (Wagner et al. 1991). Accordingly, PEI is effective in gene delivery into a variety of cell types even without the addition of cell binding ligands or endosomolytic agents (Wightman et al. 1999, Zatloukal et al. 1999). The in-vivo gene transfer efficiency of these DNA/polycation complexes into tumors also has been assessed (Kircheis et al. 1999). PEI possesses DNA binding and condensing activity, together with a high pH buffering capacity, that is believed to protect DNA from nuclease degradation and to enhance its exit from the endosomal compartment (Bieber et al. 2002, Pollard et al. 1998). Every third atom of the PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH (Boussif et al. 1995). The intracellular transport pathway from the endosome to the nucleus is still not well understood. In this research, the higher gene transfer efficiency of the PEI-SV method, and the detection of reporter gene in the sperm cells, revealed that PEI is also effective in delivering genes into abalone sperm cells. The mechanism is presumably that the PEI/DNA complex is taken up by endocytosis and then carried into the eggs by the sperm cells. Whether DNA is integrated into the nucleus of the sperm cells before or after fertilization is under further investigation.

In the PEI group, the result showed that PEI decreased the survival rate and the gene delivering efficiency was low, which was presumably because the chorion of the abalone fertilized eggs blocked the PEI/DNA complex from entering. The mosaic expressions of GFP suggest that the PEI can deliver genes into one or more cells rather than into all cells of the embryos at two or more cell stages. The fertilization rate and survival rate of the PEI-SV group were significantly lower than the control group, which is presumably because of the lower motility of the sperms caused by the viscosity or cytotoxicity of the PEI. Recently, for its DNA-condensing and pH-buffering properties, PEI has been widely used as a highly efficient nonviral vector in gene therapy for delivering oligonucleotides and plasmids both in vitro and in vivo (Putnam et al. 2001, Kircheis et al. 1999). However, it was found that high concentrations of PEI/DNA complexes (>6 mg/mL) induced a cellular toxic response in cultured mammalian cells (Boussif et al. 1995). In this study, the lower fertility rate and survival rate of the PEI group and the PEI-SV group, compared with the control group, showed that PEI may be cytotoxic to the abalone sperm cells or embryo cells. Nevertheless, the PEI-mediated sperm-vector method is far more efficient and convenient compared with the conventional methods, and is potentially a powerful tool in transferring genes into other shellfish, fish, and even mammalian species.

TABLE 1.

Fertility rate, survival rate and GFP expression data in each
group (%).
                                                            GFP
                  Total     Fertilization    Survival    Expression
    Group         Number         (%)           (%)          (%)

Control           50,000        97.4           83.6          0
Microinjection       500        97.4            0            0
SV                10,000        95.2           82.3          0
PEI               10,000        97.4           52.7          3.8
PEI-SV            10,000        16.3            7.6         13.9

ACKNOWLEDGMENT

This work was funded by the national High-tech Youth foundation project (2001AA628050, 2004AA603830) and national key fundamental research and development project (G1999012009).

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XIAOLONG WANG, JINGJIE HU, JIE PAN, ZHUOJUN MA, KE BI, QUANQI ZHANG, AND ZHENMIN BAO *

Laboratory of Marine Genetics and Breeding, Department of Bioteehnology, Ocean University of China, Qingdao 266003, People's Republic of China

* Corresponding author. E-mail: [e-mail]