New gene transfer methods

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NEW GENE TRANSFER METHODS R. J. Wall Gene Evaluation and Mapping Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20750 ABSTRACT The intentional introduction of recombinant DNA molecules into a living organism can be achieved in many ways. Viruses have been making a living by practicing gene transfer for millennia. Recently, man has gotten into the act. The paradigm employed is fairly straightforward. First, a way must be found to move genetic information across biological membrane barriers. Then, presumably, DNA repair mechanisms do the rest. The array of methods available to move DNA into the nucleus provides the flexibility necessary to transfer genes into cells as physically diverse as sperm and eggs. Some of the more promising alternative strategies such as sperm-mediated gene transfer, restriction enzyme-mediated integration, metaphase II transgenesis, and a new twist on retrovirus-mediated gene transfer will be discussed, among other methods. PuMishsd by Elsevier Science Inc.

Key words: transgenic, animals, livestock, genetic engineering, GM0 INTRODUCTION For the purposes of this review, “new methods” will be defined as those developed since I last addressed this group in 1996 (75). Furthermore, this discussion will be restricted to gene transfer methods intended for production of germline transgenic animals: those animals produced with the intention of passing their transgene to subsequent generations. Somatic cell gene transfer (gene therapy), a vibrant field of scientific investigation, will only be mentioned briefly. My topic of discussion in 1996 was gene transfer by pronuclear microinjection. The current topic of discussion is nearly everything else, except for possibly the most tantalizing new method available to livestock species-gene targeting via nuclear transfer (14, 47, 53). That topic will be discussed by Prof. Jean-Paul Renard elsewhere in these proceedings. Gene transfer, in the context of this discussion, involves two distinctly separate processes. The first step in the process must provide a mechanism by which genetic information can be transported from extracellular space, across biological membranes, and into the nucleus so that the incoming genetic information co-mingles with the genome of the target organism. The second step in the process affords a means for the new genetic information to become part of the target genome. Most efforts to enhance gene transfer have focused on finding new ways to move transgenes across barriers or into new “vehicles” to assist in carrying the transgenes across barriers. Therefore, most of this review is about the first step, though a few interesting new approaches for improving integration frequency are also discussed. Pronuclear microinjection was the first gene transfer technique designed specifically to produce germline transgenic animals. However, pronuclear microinjection was not the first gene Theriogenology 57:169-201, 2002 Published by Elsevier Science Inc.

0093-691W02/$-see front matter PII: SOO93-691X(01)00666-5

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transfer technique employed to introduce foreign DNA into embryos. The first such attempt seems to have utilized a fairly unconventional approach, even by today’s standards-spermmediated gene transfer (4). A few years later, Rudy Jaenisch and Beatrice Mintz used Mother Nature’s gene transfer vector, the virus, to introduce new genes into blastocysts. They demonstrated that the viral genes persisted in adults and elicited a phenotype (30, 3 1). Though this manuscript is not about pronuclear gene transfer, it might be useful to briefly list the characteristics of that methodology for comparison purposes. Possibly the most important characteristic of gene transfer by pronuclear microinjection is its species independence, at least in mammals. Pronuclear microinjection has successfully generated transgenic animals in a wide variety of mammalian species: mice (23), pigs, sheep, and rabbits (25), rats (24, 48), goats (17) and cattle (3, 26). In addition to its versatility, pronuclear microinjection is also one of the most straightforward methods. Preparation of the transgene requires little beyond what is required to build the construct (linearize and optionally separate from vector backbone). The embryo manipulation required is challenging, but not more challenging than that required by other protocols. Its versatility and simplicity are its most compelling features; its low efficiency is, without question, its greatest deficiency. The low efficiency is attributable to poor embryo survival to term, low transgene integration rates, and unpredictable transgene behavior. Most of the alternative gene transfer strategies are designed to overcome one or more of these inadequacies. There are a myriad of ways to transfer genetic material into an organism. No one technique embodies all of the most desirable attributes. Therefore, the choice of the “best” gene transfer method is dependent on the goals and hurdles imposed by a particular project. Transferring genes into whole organisms presents different challenges than transferring genetic material into cells, thus dictating different strategies. This review will not deal specifically with cell culture transfection methodology. There is, however, a significant overlap in approaches between those distantly different goals. Indeed, the majority of techniques for producing transgenic animals were first evaluated in cell culture. To select the most efficacious technique, it is useful to define project goals and establish criteria by which alternative methods can be judged. Generally, the goals of transgenic animal genes of interest, modulating gene experiments have been limited to “over-expressing” expression, “knocking out” expression of genes, or altering the primary structure of an endogenous gene product. Pronuclear microinjection has successfully been employed to achieve The latter two goals require “gene over-expression and modulation of gene expression. targeting” approaches. Though claims have been made for successful gene targeting by pronuclear microinjection in mice (61), rigorous proof has not been documented in peerreviewed scientific journals to my knowledge. In practice, until just a few years ago, most transgenic animal projects either used pronuclear microinjection or ES cell-mediated chimera production. In part motivated by the desire to find a more efficient way of making transgenic livestock, somatic cell nuclear transfer was developed (77). Between the development of gene transfer strategies in the 1980s (23, 71) and somatic cell nuclear transfer, a number of gene transfer techniques have been developed and refined. Some of the more interesting and/or promising techniques are discussed here.

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NEW GENE TRANSFER TECHNIQUES Retrovirus Viral-mediated gene transfer is possibly the original asexual gene transfer approach for mammals and, as mentioned, one of the first gene transfer approaches tried as a laboratory technique. Therefore, it does not seem to qualify as a new technique. However, a clever new modification to this approach merits inclusion in this discussion. Viral-mediated gene transfer has been successfully employed in a wide range of species: chickens (7, 60), medaka (39), surfclams (40), and zebrafish (38), in addition to the original application in mice (69). More recently, retrovirus-mediated gene transfer has been used to produce transgenic cattle (12) and to produce transgenic rhesus monkeys (11). The new retroviral-mediated gene transfer’s effectiveness was clearIy demonstrated by Chan and Bremel when they reported the production of transgenic cattle (12). In that study, four of four calves born were transgenic using their novel treatment of infecting metaphase II (MB) stage oocytes. One hundred percent efftciency is an extraordinary result for a transgenic cattle project. No transgene expression data were presented. Based on previous viral gene transfer studies in the mouse, one might expect variegated expression in some of those calves. The primary advantages of this approach are high frequency of gene transfer across embryonic membranes, high integration into oocyte/zygotic genome, and minimal required embryo manipulation. These characteristics result in high transgenesis efficiency, arguably the most efficient method available. So why haven’t all switched to using retrovirus vectors for transgenic livestock production? Retrovirus-mediated gene transfer has some disadvantages (76). One of the most serious limitations of retrovirus gene transfer is the relatively small amount of genetic information (~10 kb) that can be transported because of the physical limitation in volume of the viral particles. This is a potential problem because, to ameliorate variable expression of transgenes caused by the so-called “position effect,” investigators have been tending to use longer regulatory sequences and have been increasing the length of surrounding flanking sequences. To reduce sequence length for use in retrovirus vectors, transgenes have been placed under the control of the virus’s long terminal repeats (LTRs), the flanking sequences of the provirus genome. That strategy can be responsible for the second flaw of retroviral vectors. In some experiments, transcription of transgenes has been reduced when under LTR control or transgenes can become hypermethylated in the context of LTRs (57). Using mutated LTRs or using regulatory elements of mammalian origin, at the expense of lengthening the transgene, may overcome gene expression limitations (68). A third potential disadvantage of retrovirusmediated gene transfer is the complexity of the process. Though “introducing” viral particles to oocytes requires the least complicated embryo manipulation, packaging transgenes into virions takes many steps. Transgenes must be built, assembling regulatory elements, coding, signal peptide, and polyadenylation signal sequences, as for any gene transfer approach. Then the transgene must be introduced into the proviral genome by standard molecular cloning techniques. The modified proviral genome is then transfected into packaging cells, and the

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packaging cells are grown to produce viruses. used to infect oocytes or embryos.

Once the viruses are concentrated, they can be

The potential significance of transgenic rhesus monkeys (11) for the biomedical community is obvious. The fact that transgenesis in the monkey was finally accomplished using retroviral-mediated gene transfer attests to the power and versatility of that approach. Even so, most laboratories involved in production of transgenic livestock have not embraced this technology. The hesitancy on the part of those researchers to use retroviral-mediated gene transfer may be, in part, because of concerns about public perception. There are also likely to be lingering concerns of the potential consequences of recombination events between the viral vectors and endogenous retroviruses generating new pathogenic agents. This latter concern could be evaluated in rodent model systems. Dealing with public perception requires tools that technology is ill-equipped to provide. Sperm-Mediated Gene Transfer What could be more straightforward than producing functional transgenic animals by simply dipping sperm in a solution of transgenes and then using those sperm for artificial insemination? The field of sperm-mediated gene transfer has been plagued, in the past, by lack of rigorousness by some critics and some proponents. Following the first demonstration of sperm-mediated gene transfer (4), few seemed to take note. However, Marialuisa Lavitrano’s Cell paper (36), describing the production of transgenic mice by sperm-mediated gene transfer, generated a flurry of interest. Initial attempts to repeat Lavitrano’s work were unsuccessful (6). Nevertheless, a number of intrepid investigators, encouraged by the results of basic studies into the mechanism of sperm-mediated gene transfer led primarily by Corado Spadafora, continued to Spadafora, in explore the possibility of using sperm to carry transgenes into oocytes. collaboration with others, has generated a body of work that helps explain how sperm-mediated gene transfer might work, in addition to revealing some interesting and novel aspects of sperm physiology (22, 41, 43, 44, 52, 63, 70, 79, 80). Sperm-mediated gene transfer has now been demonstrated in a wide variety of species: cattle and chicken (66), golden hamster (18), pig (8, 55,63), rabbit (35), salmon (67), shell fish (73), silkworm (65), frog (32), and zebrafish (34). The most apparent advantage of sperm-mediated gene transfer is its simplicity and the minimal embryo handling required. However, the previously cited examples of sperm-mediated gene transfer are not without limitations. All of these cited studies demonstrated successful gene transfer, with varying degrees of thoroughness, but few of the studies convincingly demonstrate transgene expression. It is likely that sperm-mediated gene transfer protocols will continue to be refined. If reliable transgene expression can be achieved, use of sperm-mediated gene transfer In species in which may rival the popularity of pronuclear microinjection in a decade. manipulation of oocytes or zygotes presents a particularly difficult challenge, sperm-mediated gene transfer may turn out to be the method of choice. Valued Added Sperm-Mediated Gene Transfer Methods Although the body of evidence is still not sufficient to warrant elevating sperm-mediated gene transfer to the status of pronuclear microinjection, ES-cell chimera production, or even

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transgenesis by nuclear transfer, researchers continue to invest resources into enhancing the performance of the technique. Three studies that combine sperm-mediated gene transfer (SMGT) with other methodology are a case in point (55,59,66). Scientists in Israel have combined restriction enzyme-mediated SMGT + REMI. integration (REMI) with SMGT to produce transgenic cattle and chickens (66). Their transgenic efficiency in cattle was an impressive 100% (4/4 calves born) and an equally impressive 90% in chickens (1709 chicks). These preliminary findings are very intriguing. Once the transgenic calves produce offspring, the investigators demonstrate transgene-genome junction fragments and formally confirm transgene expression; the characteristics of this interesting refinement of SMGT will become evident. SMGT + Electroporation. Marc Sirard’s laboratory, often at the cutting edge of technology, has proposed electroporating DNA “into” sperm before performing SMGT (21). This laboratory also combined electroporation of sperm with an attempt to enhance integration efficiency by homologous recombination (59). These studies demonstrated that electroporation increases sperm-transgene interaction. The techniques used for evaluating success were able to detect the presence of the transgene, but were not sufficiently robust to convincingly demonstrate transgene integration in the resulting embryos produced. SMGT + mAb. Another recent study by investigators at BioAgri Corp. and UCLA has reported the successful production of transgenic pigs using an antibody to associate their transgene with sperm before surgical oviduct insemination (13, 55, 56). Their transgenesis rate was 20%, and they presented convincing evidence of transgene integration, expression, and transmission of the transgene to a subsequent generation. It appears that these data have been presented only in poster format to date. Publication of this work in a peer-reviewed journal should make for interesting reading. SMGT + ICSI. A technique related to SMGT utilizes intracytoplasmic sperm injection (ICSI) to transfer transgenes into oocytes. At about the same time that Teruhiko Wakayama, working in Ryuzo Yanagimachi’s laboratory, was perfecting somatic cell nuclear transfer in the mouse, Tony Perry, working in the same laboratory, was inventing a new gene transfer method (51). He called his new approach “mI1 transgenesis.” The technique involves washing caudae epididymal sperm, mixing the sperm with a solution containing transgenes at a concentration of 5 to 10 ng/uL for 1 min, mixing the sperm with PVP (polyvinylpyrrolidone) to a final concentration of 10% PVP, and performing ICSI within an hour of the initial sperm-DNA mixing. Of the 57 pups produced in that study, 19% were transgenic. The transgenesis rate reported is within the range one might expect for pronuclear microinjection. Interestingly, Perry and his colleagues found that it was necessary to pretreat sperm with Triton-x or expose them to a freeze-thaw cycle to produce transgenic offspring. Untreated sperm exposed to DNA prior to ICSI resulted in no transgenic offspring. This likely provides clues as to the mechanism by which the sperm are “carrying” transgenes into oocytes. Epididymal sperm were used in the Perry study. This, no doubt, was a matter of convenience, as ejaculated mouse sperm are not easily harvested. It remains to be determined

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whether epididymal sperm are required. Such is not likely to be the case. If epididymal sperm were required, it would be a drawback for the implementation of mII transgenesis for many livestock species. On the surface, the most significant disadvantage of this technique is the skill required to perform ICSI. Many laboratories perform ICSI routinely, so this technique should be readily transferable. Yanagimachi’s laboratory, pioneers in the development of ICSI, is well known for impressive embryo manipulation skills. Sometimes other laboratories find their manipulations challenging to replicate, at least initially. Further refinements of this approach may well be able to increase the proportion of transgenics produced. The status of mII transgenesis will be increased when other laboratories adapt this methodology. Sperm Transfection In Vitro and In Vivo Gene transfer techniques used in gene therapy and the subject of this review share many common goals, such as developing methodology for transporting transgenes across membrane barriers and stimulating insertion of the transgenes into the genome of the target cells or organism. However, the most fundamental goals of somatic cell gene transfer (gene therapy) and germline transgenesis are, as their names imply, significantly different. At least one area of investigation somewhat blurs the distinction between the two goals. Researchers have been exploring the possibility of transfecting spermatogonia in situ via infusion of transgenes into seminiferous tubules (27, 78) or transfection of germ cell precursors in vitro followed by transplantation into host testis (5). Brinster and Avarbock demonstrated that testis-derived cells transplanted into the testis of infertile males could populate the host testis, generate sperm, and produce offspring. Presumably the next step would be to transfect the testisderived cells before transplantation (49). It is not surprising that an in situ approach of infusing transgenes directly into seminiferous tubules would fail (78). Considering the design of the testes, it is hard to imagine an infusion technique that could transfect a significant proportion of developing spermatozoa. Therefore, without some additional enrichment strategy, one would expect only a very small percentage of ejaculated sperm from seminiferous tubule-infused males to carry transgenes. Huang and colleagues (27) used such an approach. They infused seminiferous tubules with transgene and then used electroporation to encourage transfection. Testes were then harvested, and “transgenic sperm,” expressing yellow fluorescent protein (YFP), were selected and used for ICSI. Transgenic pups that were YFP positive were produced. It is not clear what advantages this technique might have over other approaches described here. A slightly less cumbersome approach involves infusing transgenes into the vas deferens of a “carrier” male and then using him for natural mating a day later (28). Four apparently mosaic transgenic pups were identified out of 53 pups born using this approach. One could possibly argue that less labor is involved in this technique than with approaches that require handling each embryo.

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REM1 to Improve Integration Restriction enzyme mediated integration, first demonstrated in yeast, can enhance integration rate almost 20-fold (62). The procedure simply involves mixing a restriction endonuclease with a transgene before transfection. Presumably, the restriction enzyme maintains free ends of the transgene, which are therefore available to interact with the genome or increase the number of “useful” DNA breaks into which the transgene can integrate, or otherwise stimulates DNA double strand break repair mechanisms. Subsequent studies in yeast have shown that in addition to BarnHI, used in the original Schiestl and Petes study (62) BglII and KpnI can also increase integration rates of foreign DNA in yeast. However, many other restriction enzymes had no effect on integration (45). Since then, REMI has been shown to be an effective enhancer of transgene integration in fungi (42), protozoa (2, 2, 72), and frogs (46, 50). Therefore, it is not surprising that researchers would combine REM1 with pronuclear microinjection (64). Restriction enzyme-mediated integration did seem to increase integration efficiency (18% vs 9% transgenic mice per pup born). This observation and unpublished anecdotal evidence suggest REMI probably does increase integration efficiency of pronuclear microinjection, though the effect may be small. There are no suggestions in the current body of literature (mostly in cultured cells) that REM1 has any identifiable disadvantages. Replication of this approach in other labs will help reveal the usefulness of REM1 as a tool for enhancing transgene integration rates. Miscellaneous Techniques Several techniques are grouped in this section because they do not tit elsewhere in this discussion and because there is an insufficient body of literature to warrant an individual section for any of these methods. Nevertheless, they are included for completeness and because some appear to be potentially useful and may warrant further attention by others. Liposomes. Investigators have used cationic liposomes (Fugene, Boehringer-Manheim) to transfect mouse oocytes, which were subsequently capable of being fertilized and resulted in production of zygotes, morula, and blastocysts (9). Transgene expression was detected in the transfected embryos. The results clearly indicate that if the zona is permeabilized or removed, liposomes function as they do for other cell types to augment movement of DNA across lipid bilayers. Unfortunately, no information was provided attesting to the ability of those transfected embryos to produce live offspring. Homologous Recombination-Assisted Integration. Researchers have added sequences to the ends of their transgenes that are homologous to highly repetitive elements in the genome in the hopes of improving integration rate (33, 58). As much as a four-fold increase in integration rate has been claimed. Unfortunately, the results of both of these studies are based on PCR or gene expression in preimplantation embryos. It is, of course, difficult to distinguish between integrated and unintegrated transgenes using these techniques. The HR-assisted integration concept awaits the production of fetuses or offspring that can be subjected to more rigorous evaluation criteria.

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Carrier Proteins. Mixing a transgene with a nuclear localization signal peptide prior to injection into zebra&h egg yolks, Liang et al. achieved transgenesis rates of 21 to 94% when injecting 10 to 10,000 copies, respectively (37). Transgenesis efficiency was transgene dosedependent. However, injecting many copies to achieve high efficiency concerned the authors because it also increased the number of multiple sites of integration, possibly at sites, which, when disrupted, could be lethal. Judicious use of this attribute could be beneficial if one could regularly produce founders with a moderate number of integration sites that would segregate in subsequent generations and thus establish multiple transgenic lines. Others have also employed proteins to “carry” transgenes into embryos. In a very complex scheme, transgenes were linked to insulin via a polylysine peptide and to streptavidinbiotinylated adenovirus (29). The insulin moiety was intended to foster receptor-mediated endocytosis and the adenovirus’ role was to lyse the internalized endosome. This strategy proved very successful at transfecting embryos, but seemed to provide no enhancement in the production of transgenic mice. Large Vectors. This is a topic that has received sufficient attention in the scientific community to warrant its own section. However, an excellent review on transgenes contained in artificial chromosomes has recently been published (15). Further discussion here would be Transfer of large, chromosome-sized pieces of DNA does not seem to alter redundant. transgenesis efficiency, but it does seem to provide an effective way of circumventing the unpredictable behavior of transgenes, attributable to the position effect. The studies reported in the Co review (15) were in essence treating artificial chromosomes as gigantic plasmids. But what if these very large pieces of DNA were designed to function as independent chromosomes? That is exactly what has been reported in a preliminary study based on murine satellite DNA-based artificial chromosomes (SATAC). These SATACs are 60- to 400megabase chromosomes (16). This single report of the use of SATACs to produce transgenic mice by pronuclear microinjection clearly demonstrates SATACs can be transmitted to Fls and remain episomal. It is too early to tell whether this approach will have a significant impact on the field of transgenic animals. It is a technology definitely worth watching. FINAL THOUGHTS I have purposely neglected somatic cell gene transfer (gene therapy) studies because I considered them beyond the scope of this review’s main topic. Anyone seriously interested in exploring alternative methods to enhance the efficiency of producing germline transgenic animals might want to review that literature periodically. Most of the gene therapy vectors are viral-based. However, there are other approaches that involve shooting genes into tissues and organs (10, 19, 20). It has been difficult to find many compelling agricultural applications for the gene therapy approach primarily because it is likely that most foreign proteins produced by inserting a new gene into somatic tissue would illicit an immune response. Evaluating gene constructs intended for expression in tissues for which robust tissue culture systems do not exist, such as mammary gland tissue, might be one of this technology’s applications in an agricultural context. Of course, an immune response would be ideal if one was trying to develop a vaccine (74), possibly for foot & mouth disease (1) or anthrax (54).

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I was surprised to see how much space I have devoted to sperm-related gene transfer methods in this review. That was not my intention when I began reviewing the literature prior to writing this document. I do have to admit a bias in favor of techniques that achieve one’s goals with minimal effort. By definition, SMGT would seem to fit that paradigm, at least in principle. However, I attempted to report findings in proportion to what is currently being published. It seemed to me that a relatively high proportion of innovative gene transfer methods involve using sperm as a transgene transport system. The only conclusion I am able to draw at the moment is that SMGT methods are a popular topic of investigation. However, SMGT is not a “main stream” method for producing transgenic animals, at least not yet. REFERENCES 1.

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