Sperm-mediated transgenesis

The idea of using a sperm cell for introducing exogenous DNA into an oocyte at the time of fertilization would be brilliant if only we were sure that it can be done. Since 1989, contradictory reports have appeared in the literature and, at present, no consensus has been reached on the topic. Given the potential impact of this method for the generation of transgenic animals, for both mammalian and non-mammalian species, this review summarizes what has been achieved in this field. While some aspects, such as the binding of DNA molecules to spermatozoa, have now a solid experimental base, others, such as the generation of real transgenic individuals, are still based on disputed evidence. A critical analysis of the most relevant data will be presented in order to provide the tools for an objective evaluation of the efficiency of this method.     

精子介导的转基因技术

精子介导的转基因技术是近十几年发展起来的一种新技术.从各个种的实验证明,精子有瞬间吸收外源DNA的能力.这一过程由一系列因子起重要调节作用.一种特异的DNA结合蛋白介导了外源DNA与精子的结合,同时精浆中存在一种因子起抑制两者结合的拮抗作用.CD4分子与外源基因的内化有关.内化的DNA可能与精子核支架结构(nuclear scaffold)结合,并整合或重排.但仍需要大量实验数据进一步证明是否产生真正可遗传的转基因后代,及如何提高转基因效率,以使这一方法得到普遍的推广及应用.     

The making of "transgenic spermatozoa"

The processes of making transgenic animals by microinjecting DNA into the pronucleus of a fertilized oocyte or after the transfection of embryonic stem cells are now well established. However, attempts have also been made, with varying degrees of success, to use spermatozoa as a vector for transgenesis in mammals and other vertebrates during the last decade. A number of different approaches for making transgenic spermatozoa have been developed. These include directly incubating mature, isolated spermatozoa with DNA or pretreating mature, isolated spermatozoa before assisted fertilization. Microinjection procedures have also been established to transfect male germ cells directly in vivo within the seminiferous tubules or to reimplant previously isolated male germ cells submitted to in vitro transfection into a recipient testis. The latter two techniques present the advantage of being able to create transgenic progeny simply by mating with wild-type females, which avoids the possibility of interference or damage as a result of assisted fertilization or the manipulation of embryos. The different aspects of sperm-mediated transgenesis are presented.     

Spontaneous uptake of exogenous DNA by bull spermatozoa

Sperm-mediated DNA transfer can be used to transfer exogenous DNA into the oocyte for the production of transgenic animals. In spite of controversy in the literature, sperm-mediated DNA transfer is a simple and quick technique that can be used in routine breeding programs (AI, embryo transfer and IVF). The main objective of this study was to determine the factors affecting the spontaneous uptake of exogenous DNA by bull spermatozoa. For this purpose, fresh and frozen spermatozoa (0.25 x 10(6)), from the same ejaculate from each of four bulls were co-incubated with fluorescent-labeled green fluorescent protein (GFP) and chloremphenicol acetyltransferase (CAT) plasmids at 37 degrees C for 30 min. Neither bull nor plasmid significantly affected the uptake of exogenous DNA. However, transfection efficiency was higher in frozen-thawed versus fresh spermatozoa (P<0.001). Regardless of whether transfected spermatozoa were alive or dead, all transfected spermatozoa were immotile. It can be concluded that a population of spermatozoa is present in bull semen which has the ability to uptake exogenous DNA spontaneously. There is tremendous scope to improve transfection efficiency of spermatozoa while maintaining motility; this needs to be achieved in order to more easily use this technique in transgenesis. However, live-transfected bull spermatozoa clearly can incorporate exogenous DNA and should be usable in intracytoplasmic sperm injection protocols.     

精子介导转基因动物的制备

近年来 ,通过显微注射DNA至孵育的卵母细胞原核或外源基因转染后的胚胎干细胞进行转基因动物的生产已取得了令人瞩目的成就。在过去的 1 0年中 ,以精子作载体制备转基因哺乳动物或脊椎动物也取得了一些不同程度的进展。这些技术主要包括 :直接将外源DNA与精子共孵育至成熟 ;提取分离的精子DNA或进行预处理至精子发育成熟 ;以及在辅助受精前分离精子细胞等。此外 ,一些显微注射技术 ,如在输精管内进行体内直接转染雄性生殖细胞 ;将体内转染的雄性生殖细胞植入已分离的雄性生殖细胞 ,再显微注射至受体的睾丸 ,这些技术也逐渐成熟起来。研究表明 ,通过体内、体外转染外源DNA的显微操作技术只需将雄性受体与野生型雌性交配就可产生出转基因的后代个体 ,同时也避免了辅助受精和胚胎操作带来的机械损伤 ,因此具有一定的优势。本文综述了精子介导转基因 (SMGT)技术的发展历程、研究现状及前沿进展。     

Sperm-mediated gene transfer into oocytes of the golden hamster: assessment of sperm function

The possibility of sperm as a vehicle to deliver foreign DNA to oocytes was tested in hamsters. Epididymal spermatozoa, incubated with linearized plasmid DNA encoding ovine growth hormone (pCMXoGH), showed a spontaneous tendency to interact with DNA. Kinetics of sperm uptake of DNA was determined by using [32P]-labeled DNA. Spermatozoa took up the added DNA by 15-30 min and the uptake was inhibited by human seminal fluid in a dose dependent manner. Addition of DNA did not affect the functional competence of spermatozoa, in terms of their ability to undergo capacitation and acrosome reaction (34.5% +/- 2.2 vs 35% +/- 1.5). The fertilizing ability of DNA treated-spermatozoa from hamsters and humans was assessed by zona-free hamster egg penetration assay. Number of sperm penetrated per oocyte were 23 +/- 4.5 and 1.4 +/- 1.3 for hamster and human spermatozoa, respectively. Penetrated oocytes harbored sperm-treated DNA both with hamster (30.2 cpm/oocyte) and human (19.2 cpm/oocyte) spermatozoa. These results show that the hamster and human spermatozoa have a strong tendency to interact with exogenous (foreign) DNA and are able to transfer DNA to oocytes. Sperm may be used as a vector for DNA transfer and this approach has potential in the production of transgenic animals.     

Association of rabbit sperm cells with exogenous DNA

The ability of rabbit spermatozoa to bind exogenous DNA during sperm-mediated gene transfer (SMGT) was tested in our study. Fresh collected semen, or fully capacitated sperm cells, was co-cultured with plasmid DNA labeled with tetramethyrodamine-6-dUTP. Fluorescent spermatozoa were counted before and after DNaseI treatment. Results showed that fluorescent-labeled plasmid DNA could be taken up by capacitated rabbit sperm cells. 66% spermatozoa carried exogenous DNA in the presence of lipofectin. Bovine serum albumin could block this process effectively. Associated DNA was mainly located in the posterior area of the sperm head. In order to verify whether exogenous DNA was carried into the embryo and expressed in the offspring, further SMGT experiments were carried out using the pHM-CR plasmid which contains LacZ and Neomycin genes. beta-galactosidase was expressed in different stages of embryo development and in the tissues of young rabbit as detected by using X-gal staining. Large portion of embryos survived under the selection pressure in G418 containing medium, after SMGT. Transgene integration was further verified by PCR analysis. These results confirmed the ability of rabbit sperm cells to carry transgene into the embryo during in vitro fertilization.     

The negative effects of exogenous DNA binding on porcine spermatozoa are caused by removal of seminal fluid

Sperm-mediated gene transfer (SMGT) might become the most efficient and cost effective technique to generate transgenic animals, which will significantly increase their application in biomedical research and in commercial production. Despite some successes, the technique has remained controversial for almost 20 years and despite number of studies the reasons for poor reproducibility of this promising technology has not been understood. We suggest that the reason for poor reproducibility is the presence of natural defences against exogenous DNA invasion acting in spermatozoa or in embryo. Based on previous reports we have investigated the effect of foreign DNA binding on spermatozoa by monitoring motility, viability and genomic DNA damage. Evaluation of DNA binding in sperm collected from 16 boars demonstrated that 28-45% of the added pEGFP plasmid was bound to spermatozoa with 9-32% being internalized in sperm nucleus. In agreement with previous reports, our results demonstrated that the pEGFP-treated sperm show an average a 2-fold decrease in motility (p<0.05), 5-fold decrease in progressive motility (p<0.05), and 1.4-fold increase in number of sperm with highly damaged DNA (p<0.05) as detected by Comet assay. In contrast with previous reports, we demonstrate that all such changes were associated with the removal of seminal plasma during the washing step and not with foreign DNA binding per se. We suggest that poor reproducibility of SMGT most likely result from selection against DNA-loaded sperm at later stages of fertilization.     

The role of small molecules in sperm capacitation

Mammalian spermatozoa released into an appropriate environment in vitro can capacitate but then may undergo spontaneous acrosome reactions. Since successful sperm interaction with the zona pellucida of an unfertilized oocyte requires an intact sperm plasma membrane, spontaneous acrosome loss is biologically undesirable because it renders spermatozoa non-fertilizing. Several small molecules (fertilization promoting peptide [FPP], adenosine, calcitonin and adrenaline), found in various body fluids including seminal plasma, have been shown to regulate capacitation in vitro. They initially accelerate capacitation but then inhibit spontaneous acrosome loss, allowing spermatozoa to maintain their fertilizing potential. Specific receptors for all these molecules are present on mammalian spermatozoa and their activation by the appropriate ligands leads to modulation of membrane-associated adenylyl cyclase activity and production of cAMP, stimulating cAMP production in uncapacitated cells and inhibiting it in capacitated cells. Boar spermatozoa have been shown to respond in vitro to adenosine and FPP, suggesting that the addition of these molecules to sperm samples used for artificial insemination could be beneficial in helping spermatozoa maintain fertilizing potential until they reach their target.     

Liposome-mediated DNA uptake by sperm cells.

To investigate the potential use of sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method.     

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.     

外源基因对精子的影响及其在山羊早期胚胎中的表达

摘要: 在前期实验中发现, 山羊精子可自发结合外源DNA, 但结合能力在不同动物个体之间差异显著。挑选结合能力明显不同的3只公羊, 进一步探讨了外源DNA对精子的影响及其在早期胚胎中的表达, 结果发现: 外源基因与精子共同孵育后, 精子的活率、顶体反应发生率和受精能力均呈下降态势, 其降幅与精子的结合能力密切相关。利用与DNA共育后的精子进行体外受精, 外源基因可被导入卵母细胞并在早期胚胎中获得表达, 但胚胎阳性率因精子供体不同而差异显著(P<0.05); 其中来源于高、中结合能力供体生产的胚胎, 分别有16.2%(25/154)和5.3%(4/76)可检测到外源基因存在, 但表达仅见于高结合能力供体生产的早期胚胎, 表达率为6.5%(10/154); 低结合能力供体生产的胚胎无外源基因。研究表明, 在以精子载体方法生产转基因动物的实验过程中, 筛选对DNA结合能力较强的精子供体是提高转基因效率的前提, 但需要考虑外源DNA对精子受精能力的影响。     

DNA uptake in swine sperm: Effect of plasmid topology and methyl‐beta‐cyclodextrin‐mediated cholesterol depletion

Sperm‐mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real‐time PCR‐based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl‐β‐cyclodextrin (MCD)‐treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 79: 853–860, 2012. © 2012 Wiley Periodicals, Inc.     

Active integration: new strategies for transgenesis

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed “Active Transgenesis” to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac’s specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

Efficient production of transgenic mice by intracytoplasmic injection of streptolysin‐O‐treated spermatozoa

Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI‐based sperm‐mediated gene‐transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin‐O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT‐transgenic embryos resulted from pretreatment with 5 U/ml SLO for 30 min and co‐incubation with 1.0 ng/µl of an EGFP expression vector. By incubating spermatozoa with Cy‐3‐labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO‐treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO‐treated spermatozoa (iSMGT‐SLO) as compared to freeze‐thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT‐SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT‐SLO technique is an efficient method for production of transgenic animals for biomedical research. Mol. Reprod. Dev. 80: 233–241, 2013. © 2013 Wiley Periodicals, Inc.