Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print December 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.009340
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
68/5/1477    most recent
biolreprod.102.009340v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Celebi, C.
Right arrow Articles by Jégou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Celebi, C.
Right arrow Articles by Jégou, B.
Agricola
Right arrow Articles by Celebi, C.
Right arrow Articles by Jégou, B.
BIOLOGY OF REPRODUCTION 68, 1477–1483 (2003)
DOI: 10.1095/biolreprod.102.009340
© 2003 by the Society for the Study of Reproduction, Inc.

Minireview

The Making of "Transgenic Spermatozoa"1

Catherine Celebi4, Thierry Guillaudeux4, Pierrïck Auvray3,4, Virginie Vallet-Erdtmann4, and Bernard Jégou2,4,5

Groupe d'Etude de la Reproduction chez le Mâle,4 INSERM U.435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, Bretagne, France BioProtein Technologies,5 75013 Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 USE OF MATURE, ISOLATED...
 GENE TRANSFER VIA...
 CONCLUSION
 REFERENCES
 
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.

assisted reproductive technology, fertilization, in vitro fertilization, sperm, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
USE OF MATURE, ISOLATED...
 GENE TRANSFER VIA...
 CONCLUSION
 REFERENCES
 
The ability to manipulate gene expression by transgenesis, particularly in mammals, is one of the most significant advances made in the fields of experimental and applied biology during the last two decades. The impact of these approaches has been spectacular, opening up new possibilities for studying both the function and regulation of genes in vivo together with the mechanisms involved in normal and pathological developmental processes [1–3]. In addition, animal models that phenotypically resemble human diseases such as lipoatrophic diabetes [4], osteoarthritis [5], and Alzheimer disease [6] have been generated. This has enabled research scientists to make considerable progress in understanding the genetic and molecular mechanisms involved as well as in developing treatments and validating drug targets for human diseases [7]. This approach may also lead to the development of techniques for producing viable xenografts by generating "humanized" tissues and organs in animals such as pigs [8]. Transgenesis is therefore a key tool for the development of animals with high added value intended for pharmaceutical or industrial use, such as those producing proteins like human clotting factors [9].

Two major techniques are commonly used to make transgenic animals. The first approach, qualified as additive transgenesis, involves the microinjection of a transgene into the male pronucleus of a fertilized egg [10, 11], leading to the random insertion of exogenous DNA into the genome. When it reaches the 2-cell stage, the embryo is reimplanted into the uterus of a pseudopregnant female. Depending on the skill of the research worker, the success rate of this technique ranges from approximately 1% to 8% of the total number of microinjected eggs [12–17]. The second approach involves the in vitro transfection of embryonic stem (ES) cells [18]. These cells are then transferred into a recipient blastocyst before being reimplanted into the uterus of a pseudopregnant female. This technique is highly important, because it allows the generation of knockout mice by homologous recombination. The targeted gene can then be "turned off" by an appropriate technical procedure, such as the Cre-Lox system [19].

Other recently developed approaches include the use of a lentiviral vector to transfect a 1-cell mouse embryo [20] or gene transfer into cultured embryos by electroporation [21]. Although these techniques have been extensively and successfully developed in mice and, more recently, in rats [22], their adaptation to domestic animals such as cows remains problematic [23]. Thus, a clear need to diversify transgenesis in general exists. The difficulties encountered in developing transgenic technologies in domestic animals have recently led to the exploration of ancillary techniques, such as cloning, in sheep, goats, cows, and rabbits [24–26] and to the intensification of research using spermatozoa as vectors for transgenesis. The present review aims to address and to discuss the results and possible applications of sperm-mediated gene transfer (SMGT) technologies.


   USE OF MATURE, ISOLATED SPERMATOZOA
TOP
 ABSTRACT
 INTRODUCTION
 USE OF MATURE, ISOLATED...
GENE TRANSFER VIA...
 CONCLUSION
 REFERENCES
 
Incubation of Intact Spermatozoa with DNA Before Assisted Fertilization

The first indication that exogenous DNA could be introduced into untreated sperm was provided by Brackett et al. [27] in 1971. This group claimed that DNA labeled with tritiated thymidine passed into the head of rabbit spermatozoa during incubation. Almost 20 years later, Arezzo et al. [28] showed that both homologous and heterologous DNA penetrated sea urchin sperm and that the transgene could then be expressed in the embryos. The same year, Lavitrano et al. [29] published an article indicating that circular or linear DNA could be incorporated into mouse epididymal spermatozoa by simple incubation. They reported a mendelian transmission of the transgene in 30% of the offspring when the linear DNA form was used. However, this study was rapidly and strongly contested, particularly by Brinster et al. [30], who attempted to replicate the experiment and found no transgenic animals among the 1300 mice tested.

The skepticism regarding the value of this approach did not, however, discourage all researchers. For example, Camaioni et al. [31] showed that in boar and bull sperm, the equatorial segment of the postacrosomal region was labeled in 39%–78% of cases after incubation with tritiated plasmid DNA. This ability of mature sperm cells to spontaneously take up exogenous DNA was also confirmed by Francolini et al. [32]. Furthermore, Maione et al. [33] repeated the experiments performed by Lavitrano et al. [29] and were able to show that it was, indeed, possible to incorporate DNA into the mouse spermatozoa head. In these experiments, the frequency of DNA incorporation into spermatozoa was fairly uniform (90%), but the proportion of transgenic fetuses obtained was highly variable. Indeed, depending on the experiment, between 0% and 100% (mean, 7.4%) of the progeny was found to be transgenic by Southern blot analysis. Those authors thought that this extreme transmission disparity might be caused by specific factors of unknown origin. In fact, Lavitrano et al. [34] had previously identified a set of 30- to 35-kDa sperm proteins, conserved in many vertebrates, that specifically bind to DNA and might actively participate in the uptake of DNA by spermatozoa. Furthermore, in the same laboratory, Zani et al. [35] identified an inhibitory factor (IF-1) in the seminal fluid of different mammals as well as in sea urchin sperm that can prevent the interaction between exogenous DNA and the previously identified 30- to 35-kDa sperm proteins. The hypothesis that DNA uptake mechanisms are regulated in spermatozoa was further explored a few years later by Carballada and Esponda [36], who showed that some DNA-binding proteins are differentially expressed in mature and immature mouse spermatozoa. They also claimed that other components of the seminal plasma could exhibit an inhibitory effect.

Spadafora's work concerning the possibility of using sperm as a vector to produce genetically modified offspring [32–35] was controversial, even though the ability of sperm to carry exogenous DNA had been demonstrated with pig sperm [37]. Despite this controversy, that work prompted a number of studies in different species. In pigs, Lauria and Gandolfi [38] found that 12% of the offspring carried the transgene but that no protein was produced, which suggests a mosaical transmission. Chourrout and Perrot [39] incubated, in vain, rainbow trout spermatozoa with various DNA constructs; no transgenic fish were produced. In contrast, Khoo et al. [40] and Khoo [41] recently transmitted exogenous DNA from one generation to another via zebrafish sperm, but without the genomic integration, expression, or mendelian inheritance of the transgene. Therefore, in some cases or some species, the "transgene" may remain extrachromosomal throughout mitosis and meiosis, as has been reported in other fishes, such as loach [42]. Of note is that in the latter fish, the transgene shows a mosaic distribution in several types of tissue.

Incubation of Pretreated Spermatozoa with DNA Before Assisted Fertilization

It seems that, in the short term, the method involving the incorporation of DNA into the head of the spermatozoon, if proved to be reproducible, would be the simplest and most rapid way of producing transgenic mammals. Logically, the delivery of exogenous DNA into the head of the spermatozoon is the critical step in this approach. Even when the transgene is successfully transmitted to the offspring, the reporter gene is rarely expressed. Therefore, several teams have recently developed new procedures or used chemical treatments to improve gene transfer within spermatozoa before assisted fertilization.

Electroporation Tsai et al. [43] tested the electroporation of exogenous DNA into spermatozoa of a mollusk Haliotis divorsicolor suportexta. They used polymerase chain reaction (PCR) to show that the transgene was transmitted to the first generation in 65% of cases and integrated into the genome of some larvae. They also showed that electroporation did not affect the ability of spermatozoa to fertilize. Muller et al. [44] used a similar method in fish, notably in the common carp, African catfish, and tilapia. They found that 3%–4% of the resulting fry expressed the transgene. Finally, Patil and Khoo [45] also performed electroporation on zebrafish and showed that electroporated sperm cells took up more DNA molecules than those not subjected to the electrical field, but this technique did not increase the number of spermatozoa taking up exogenous DNA. In fact, passive incubation of spermatozoa with exogenous DNA is also sufficient to make them carriers in zebrafish. Besides, in the latter study, electroporation affected sperm motility, but fertilization rates were unaffected.

Horan et al. [46] attempted electroporation in pigs, a large mammal in which 70% of motile spermatozoa incorporated exogenous DNA in the postacrosomal region after simple incubation. The amount of DNA bound to sperm only increased by 5%–10% when this technique was used. More interestingly, Rieth et al. [47] recently showed that homologous recombination is also possible using SMGT in cattle. Indeed, transgenic embryos were obtained after electroporating sperm with DNA constructs carrying a reporter gene and a highly repetitive, Alu-like repeat known to favor transgenesis by homologous recombination. Analysis by PCR revealed that 46.5% of the transgenic embryos showed homologous recombination events, compared to only 3.5% in the absence of electroporation. Therefore, the use of this new, promising technique in addition to or instead of the use of ES cells can be envisaged in the near future to produce knockout animals, especially in large mammals.

Restriction enzyme-mediated integration In 1996, Kroll and Amaya [48] introduced linearized plasmid DNA into the nuclei of Xenopus sp. sperm by using restriction enzyme-mediated integration (REMI) to decondense the genomic DNA. The transgenic nuclei were transferred into unfertilized eggs by microinjection of sperm nuclei, and 36% of the Xenopus sp. offspring obtained were found to be transgenic, compared to 19% when the restriction enzyme was omitted from the reaction. The REMI technique is currently being used to produce transgenic Xenopus sp. and recently was applied successfully to cattle [49], in which it was combined with lipofection to integrate the transgene into the genomic DNA of the sperm before in vitro fertilization. It has also been adapted to zebrafish by Jesuthasan and Subburaju [50]. Those authors produced transgenic animals by injecting zebrafish eggs with sperm nuclei that had been submitted to various specific treatments in which the length of time that the spermatozoa were incubated with the exogenous DNA and the DNA concentrations were changed. When the conditions were suboptimal, mosaic transmission was observed.

Intracytoplasmic sperm injection after sperm/DNA interactions Other techniques involving intracytoplasmic sperm injection (ICSI) have recently been developed [51]. These methods overcome the problem associated with killing spermatozoa as a result of applying different treatments to help the exogenous DNA cross the natural sperm barrier during transfection. Thus, spermatozoa subjected to Triton X-100 treatment, repeated freeze-thaw cycles, or freeze-drying cycles before incubation with exogenous DNA [51, 52] can generate transgenic offspring. Between 64% and 94% of the blastocysts obtained carried a GFP or LacZ reporter gene, whereas only 26% of blastocysts were GFP-positive when untreated spermatozoa were used. Eight of the 11 transgenic founders generated were mated with nontransgenic mice. After mendelian transmission, 27%–50% (mean, 40%) of the offspring expressed the integrated transgene (Fig. 1A). Therefore, it clearly is possible to stably transmit a transgene in mammals using spermatozoa as a vector. Rhesus macaque embryos expressing a transgene were recently generated by this approach [53, 54].



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1. Schematic representation of the different techniques available for making "transgenic" mammals by genetically modifying male germ cells. A) In vitro transfection of spermatozoa followed by ICSI. Different permeabilization procedures of the spermatozoon plasma membrane can be used, such as Triton X-100, freeze-thawing, freeze-drying, or even REMI, before incubation with the DNA construct (step I). Oocyte fertilization is then performed by ICSI (step II), the embryos are cultured for 3 days (step III), and the viable ones are selected and reimplanted into the uterus of a pseudopregnant female (step IV). The animal models used are mostly the mouse and Xenopus sp. B) In vivo germ cell transfection via microinjection within the seminiferous tubules. The DNA to be transfected is incubated with liposomes or retroviral constructs (step I), and the cells of the seminiferous epithelium are then transfected in situ after microinjections within the seminiferous tubules or within the rete testis (step II). The cells can also be transfected via in situ or in vivo electroporation. The microinjected males are then mated with wild-type females (step III) and the progeny analyzed by PCR/Southern blotting to confirm that the transgene has, indeed, been transmitted to the offspring. The most commonly used animal model is the mouse. C) Germ cell transfection (in vitro) and transplantation (in vivo). Testes are dissected out (step I), and germ cells are prepared by mechanical and enzymatic procedures. The cells are then transfected in vitro with exogenous DNA (step II) before transplantation into aspermatogenetic mouse seminiferous tubules (step III) generated by an antimitotic treatment (busulfan) or from genetically sterile mice. The microinjected animals are then mated with wild-type females (step IV) and the progeny analyzed by PCR/Southern blotting. The most commonly used animal model is the mouse


   GENE TRANSFER VIA INTRATESTICULAR MICROINJECTION
TOP
 ABSTRACT
 INTRODUCTION
 USE OF MATURE, ISOLATED...
 GENE TRANSFER VIA...
CONCLUSION
 REFERENCES
 
DNA Microinjection

A technique consisting of microinjecting exogenous DNA directly into mouse seminiferous tubules or the rete testis (Fig. 1B) has recently been developed. This approach combines the advantages of being able to create "transgenic spermatozoa" and of being able to investigate the functions of the genes involved in spermatogenesis after the in vivo transfection of male germ stem cells. Kim et al. [55] injected a liposome/reporter plasmid complex into the seminiferous tubules of mice that had been pretreated with busulfan to deplete spermatogenesis [56]. They found that the transgene was expressed in 8%–14.8% of the tubules and used only PCR to show that it was present in some epididymal spermatozoa (7%–13%). Even though no integration could be obtained, this approach offered a new option for designing transgenic animals. Recently, our own group [57] performed the same type of experiments and analyzed the progeny of the microinjected animals. After mating these males with wild-type females, the transgene could be transmitted to the offspring but remained episomal. The plasmid was lost during the numerous cell divisions as the animals grew. Another group used the same approach, together with electroporation, to transfect cells located in the mice seminiferous tubules [58]. This resulted in long-term transgene expression in the injected testes (>2 mo).

Although this approach based on in situ DNA microinjection needs to be improved considerably before it can be used, for example, in transgenesis on large mammals, the preliminary results are encouraging. This approach can also be used as a tool in basic research laboratories to study spermatogenesis either by gene overexpression or by gene interference.

Transplantation of Genetically Transformed Germ Cells

Brinster and Zimmerman [59] as well as Brinster and Avarbock [60] have developed a technique that can potentially overcome the difficulties and uncertainties associated with direct transfection in spermatozoa. They injected preparations of freshly isolated germ cells from donor mice into the seminiferous tubules of germ cell-deficient host mice. They found that normal spermatogenesis was restored in 18%–36% of the recipients. Their experiments clearly demonstrate that it is possible to transfer freshly collected or cultured germ cells into a host mouse. Additionally, Avarbock et al. [61] succeeded in generating spermatogenesis in recipient seminiferous tubules after the transplantation of germ cells that had been frozen for 4–156 days at -196°C (Fig. 1C). The same group also attempted to develop a method for the long-term culture of stem cells [62], with the aim of eventually transfecting them in vitro before reimplantation into the seminiferous tubules of recipient testes. They successfully used a retrovirus to transfect male stem cells in vitro before microinjecting them into the seminiferous tubules [63]. The lacZ reporter transgene was expressed for more than 6 mo in the recipient mice, strongly suggesting that the transgene had been integrated in the transfected stem cells. They confirmed this hypothesis 1 yr later, when they showed the integration and the transmission of the transgene in 4.5% of the progeny, without any generation loss [64]. Brinster's group has also shown that it is possible to produce rat and hamster spermatozoa after germ cell transplantation into immunodeficient mice [65, 66]. Conversely, other attempts at xenogenic transplantation using more distantly related species were not successful [67]. Finally, Huang et al. [68] recently transfected mice male germ cells in vivo with a gene encoding the YFP fluorescent protein and obtained fluorescent spermatozoa, which were eventually used for ICSI. The transgene was then transmitted and integrated into the genome of the progeny with the selected spermatozoa.

As well as making possible the generation of transgenic animals, this gene transfer technology via intratesticular microinjection offers the possibility to perform male-sterility rescue experiments such as those recently performed successfully by Ikawa et al. [69] after transfection of Sertoli cells in situ. It also opens the way for germinal gene therapy and has the potential to produce both gametes from species on the brink of extinction and genetically modified gametes from species of great economical value.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 USE OF MATURE, ISOLATED...
 GENE TRANSFER VIA...
 CONCLUSION
REFERENCES
 
The SMGT technologies described in the present review and summarized in Table 1 provide important new perspectives in the field of animal transgenesis. However, a number of questions remain to be answered. First, large differences in transfection efficiency exist between species. Undoubtedly, the plasma membrane of the spermatozoon is one of the obstacles preventing the incorporation of exogenous DNA. Therefore, because permeabilization of this membrane is often required for gene transfer, damage incurred to the genomic DNA of the spermatozoon after permeabilization may, in some cases, have deleterious effects on the integrity of the generated animals. Furthermore, in most of the studies performed (except with cattle, in which Rieth et al. [47] successful achieved homologous recombination), the transgenes were randomly integrated into the spermatozoon genome, as in the classical technique of additive transgenesis. Therefore, a better understanding of these domains should greatly improve the efficiency of SMGT and the targeting of the transgene within the genome. Other important factors that may play a significant role are the insertional mechanisms involved in transgene integration and their effects on DNA structure. During haploid cell differentiation (spermiogenesis), the germ cell nucleus undergoes numerous modifications in which the sequential expression of histones, intermediate proteins, and protamines ultimately leads to the complex compaction of the chromatin [70]. Condensation of DNA in spermatozoa shows intra- and interspecies variations, which most probably explain, in part, the differences in efficiency observed between techniques and from one species to another. Once it has been technically refined, SMGT will certainly contribute to the development of new transgenesis methods for research and biotechnology.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Summary of the various studies on SMGT.a

Another serious concern highlighted by the data summarized in the present review is that foreign DNA can be efficiently transmitted to embryos in different mammalian species via spermatozoa. This implies that clinicians using ICSI to treat infertility should take particular precautions to exclude exogenous DNA. Contamination with foreign DNA during ICSI procedure has yet to be proven in humans. However, the presence of endogenous retroviruses (chromosomal elements with a genome organization analogous to that of exogenous retroviruses) or retrotransposons (DNA organized in a manner similar to that of retrovirus DNA) within cancerous or normal male germ cells [71, 72], most probably originating from ancient infections of germ cells and possibly involved in as-yet-unknown evolutionary aspects, shows that the introduction of exogenous DNA in the germ cell genome is more than simply hypothetical [73]. Exogenous DNA, present in the marine environment or in the soil, might participate, after incorporation, in genetic transformations in aquatic animals that have external fertilization or in bacteria, respectively [74–76]. Further support is provided by the hypothesis that DNA may play an important role as an adaptation event during evolution.

Finally, although some SMGT clearly will be extremely valuable for generating transgenic animals, genetically modified male germ cells in humans should only be envisaged for the development of gene therapy if the present international ban on germ cell genetic manipulation is lifted. It remains debatable whether such a move is desirable.


   FOOTNOTES
 
1 Supported by grants from INSERM. C.C. is a recipient of a fellowship from the Region Bretagne. Back

2 Correspondence. FAX: 33 223 23 50 55; bernard.jegou{at}rennes.inserm.fr Back

3 Current address: ONCODESIGN Biotechnology S.A., Parc technologique Toisson d'Or, BP 27627-21076 Dijon, France. Back

Received: 15 July 2002.

First decision: 5 August 2002.

Accepted: 18 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 USE OF MATURE, ISOLATED...
 GENE TRANSFER VIA...
 CONCLUSION
 REFERENCES
 

  1. Chan AWS. Transgenic animals: current and alternative strategies. Cloning 1999 1:25-46
  2. Wall RJ. New gene transfer methods. Theriogenology 2002 57:189-201[CrossRef][Medline]
  3. Mullins LJ, Mullins JJ. Transgenesis in the rat and larger mammals. J Clin Invest 1996 97:1557-1560[Medline]
  4. Reitman ML, Mason MM, Moitra J, Gavrilova O, Marcus-Samuels B, Eckhaus M, Vinson C. Transgenic mice lacking white fat: models for understanding human lipoatrophic diabetes. Ann N Y Acad Sci 1999 892:289-296[CrossRef][Medline]
  5. Goldring MB. The role of cytokines as inflammatory mediators in osteoarthritis: lessons from animal models. Connect Tissue Res 1999 40:1-11[Medline]
  6. Van Leuven F. Single and multiple transgenic mice as models for Alzheimer's disease. Prog Neurobiol 2000 61:305-312[CrossRef][Medline]
  7. West DB, Iakougova O, Olsson C, Ross D, Ohmen J, Chatterjee A. Mouse genetics/genomics: an effective approach for drug target discovery and validation. Med Res Rev 2000 20:216-230[CrossRef][Medline]
  8. Vanhove B, Renard JP, Soulillou JP. Genetic engineering in the pig. Gene knockout and alternative techniques. Ann N Y Acad Sci 1998 862:28-36[CrossRef][Medline]
  9. Niemann H, Halter R, Carnwath JW, Herrmann D, Lemme E, Paul D. Expression of human blood clotting factor VIII in the mammary gland of transgenic sheep. Transgenic Res 1999 8:237-247[CrossRef][Medline]
  10. Gordon JW, Ruddle FH. Gene transfer into mouse embryos: production of transgenic mice by pronuclear injection. Methods Enzymol 1983 101:411-433[Medline]
  11. Wall RJ. Pronuclear microinjection. Cloning Stem Cells 2001 3:209-220[CrossRef][Medline]
  12. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A 1980 77:7380-7384[Abstract/Free Full Text]
  13. Brinster RL, Chen HY, Trumbauer M, Senear AW, Warren R, Palmiter RD. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 1981 27:223-231[CrossRef][Medline]
  14. Gordon JW, Ruddle FH. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 1981 214:1244-1246[Abstract/Free Full Text]
  15. Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the Mouse Embryo—A Laboratory Manual. John Inglis: Cold Spring Harbor, NY; 1994
  16. Simoni M. Transgenic animals in male reproduction research. Exp Clin Endocrinol 1994 102:419-433[Medline]
  17. Rulicke T, Hubscher U. Germ line transformation of mammals by pronuclear microinjection. Exp Physiol 2000 85:589-601[Abstract]
  18. Capecchi MR. Altering the genome by homologous recombination. Science 1989 244:1288-1292[Abstract/Free Full Text]
  19. Lakso M, Sauer B, Mosinger B Jr, Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A 1992 89:6232-6236[Abstract/Free Full Text]
  20. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002 295:868-872[Abstract/Free Full Text]
  21. Osumi N, Inoue T. Gene transfer into cultured mammalian embryos by electroporation. Methods 2001 24:35-42[CrossRef][Medline]
  22. Charreau B, Tesson L, Soulillou JP, Pourcel C, Anegon I. Transgenesis in rats: technical aspects and models. Transgenic Res 1996 5:223-234[CrossRef][Medline]
  23. Wall RJ, Kerr DE, Bondioli KR. Transgenic dairy cattle: genetic engineering on a large scale. J Dairy Sci 1997 80:2213-2224[Abstract]
  24. Fulka J Jr, First NL, Loi P, Moor RM. Cloning by somatic cell nuclear transfer. Bioessays 1998 20:847-851[CrossRef][Medline]
  25. Dove A. Milking the genome for profit. Nat Biotechnol 2000 18:1045-1048[CrossRef][Medline]
  26. Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol 2002 20:366-369[CrossRef][Medline]
  27. Brackett BG, Baranska W, Sawicki W, Koprowski H. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci U S A 1971 68:353-357[Abstract/Free Full Text]
  28. Arezzo F. Sea urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep 1989 13:391-404[CrossRef]
  29. Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 1989 57:717-723[CrossRef][Medline]
  30. Brinster RL, Sandgren EP, Behringer RR, Palmiter RD. No simple solution for making transgenic mice. Cell 1989 59:239-241[CrossRef][Medline]
  31. Camaioni A, Russo MA, Odorisio T, Gandolfi F, Fazio VM, Siracusa G. Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads. J Reprod Fertil 1992 96:203-212[Abstract/Free Full Text]
  32. Francolini M, Lavitrano M, Lamia CL, French D, Frati L, Cotelli F, Spadafora C. Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol Reprod Dev 1993 34:133-139[CrossRef][Medline]
  33. Maione B, Lavitrano M, Spadafora C, Kiessling AA. Sperm-mediated gene transfer in mice. Mol Reprod Dev 1998 50:406-409[CrossRef][Medline]
  34. Lavitrano M, French D, Zani M, Frati L, Spadafora C. The interaction between exogenous DNA and sperm cells. Mol Reprod Dev 1992 31:161-169[CrossRef][Medline]
  35. Zani M, Lavitrano M, French D, Lulli V, Maione B, Sperandio S, Spadafora C. The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp Cell Res 1995 217:57-64[CrossRef][Medline]
  36. Carballada R, Esponda P. Regulation of foreign DNA uptake by mouse spermatozoa. Exp Cell Res 2001 262:104-113[CrossRef][Medline]
  37. Horan R, Powell R, McQuaid S, Gannon F, Houghton JA. Association of foreign DNA with porcine spermatozoa. Arch Androl 1991 26:83-92[Medline]
  38. Lauria A, Gandolfi F. Recent advances in sperm cell mediated gene transfer. Mol Reprod Dev 1993 36:255-257[CrossRef][Medline]
  39. Chourrout D, Perrot E. No transgenic rainbow trout produced with sperm incubated with linear DNA. Mol Mar Biol Biotechnol 1992 1:282-285[Medline]
  40. Khoo HW, Ang LH, Lim JHB, Wong V. Sperm cells as vectors for introducing foreign DNA into the zebrafish. Aquaculture 1992 107:1-19
  41. Khoo HW. Sperm-mediated gene transfer studies on zebrafish in Singapore. Mol Reprod Dev 2000 56:278-280
  42. Tsai HJ. Electroporated sperm mediation of a gene transfer system for finfish and shellfish. Mol Reprod Dev 2000 56:281-284
  43. Tsai HJ, Lai CH, Yang HS. Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta). Transgenic Res 1997 6:85-95[CrossRef][Medline]
  44. Muller F, Ivics Z, Erdelyi F, Papp T, Varadi L, Horvath L, Maclean N. Introducing foreign genes into fish eggs with electroporated sperm as a carrier. Mol Mar Biol Biotechnol 1992 1:276-281[Medline]
  45. Patil JG, Khoo HW. Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation. J Exp Zool 1996 274:121-129[CrossRef][Medline]
  46. Horan R, Powell R, Bird JM, Gannon F, Houghton JA. Effects of electropermeabilization on the association of foreign DNA with pig sperm. Arch Androl 1992 28:105-114[Medline]
  47. Rieth A, Pothier F, Sirard MA. Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events. Mol Reprod Dev 2000 57:338-345[CrossRef][Medline]
  48. Kroll KL, Amaya E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 1996 122:3173-3183[Abstract]
  49. Shemesh M, Gurevich M, Harel-Markowitz E, Benvenisti L, Shore LS, Stram Y. Gene integration into bovine sperm genome and its expression in transgenic offspring. Mol Reprod Dev 2000 56:306-308[CrossRef][Medline]
  50. Jesuthasan S, Subburaju S. Gene transfer into zebrafish by sperm nuclear transplantation. Dev Biol 2002 242:88-95[CrossRef][Medline]
  51. Wakayama T, Yanagimachi R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat Biotechnol 1998 16:639-641[CrossRef][Medline]
  52. Perry AC, Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y, Yanagimachi R. Mammalian transgenesis by intracytoplasmic sperm injection. Science 1999 284:1180-1183[Abstract/Free Full Text]
  53. Chan AW, Luetjens CM, Dominko T, Ramalho-Santos J, Simerly CR, Hewitson L, Schatten G. TransgenICSI reviewed: foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey. Mol Reprod Dev 2000 56:325-328
  54. Chan AW, Luetjens CM, Dominko T, Ramalho-Santos J, Simerly CR, Hewitson L, Schatten G. Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births. Mol Hum Reprod 2000 6:26-33[Abstract/Free Full Text]
  55. Kim JH, Jung-Ha HS, Lee HT, Chung KS. Development of a positive method for male stem cell-mediated gene transfer in mouse and pig. Mol Reprod Dev 1997 46:515-526[CrossRef][Medline]
  56. Bucci LR, Meistrich ML. Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutat Res 1987 176:259-268[CrossRef][Medline]
  57. Celebi C, Auvray P, Benvegnu T, Plusquellec D, Jégou B, Guillaudeux T. Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol Reprod Dev 2002 62:477-482[CrossRef][Medline]
  58. Yamazaki Y, Fujimoto H, Ando H, Ohyama T, Hirota Y, Noce T. In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation. Biol Reprod 1998 59:1439-1444[Abstract/Free Full Text]
  59. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994 91:11298-11302[Abstract/Free Full Text]
  60. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 1994 91:11303-11307[Abstract/Free Full Text]
  61. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996 2:693-696[CrossRef][Medline]
  62. Nagano M, Avarbock MR, Leonida EB, Brinster CJ, Brinster RL. Culture of mouse spermatogonial stem cells. Tissue Cell 1998 30:389-397[CrossRef][Medline]
  63. Nagano M, Shinohara T, Avarbock MR, Brinster RL. Retrovirus-mediated gene delivery into male germ line stem cells. FEBS Lett 2000 475:7-10[CrossRef][Medline]
  64. Nagano M, Brinster CJ, Orwig KE, Ryu BY, Avarbock MR, Brinster RL. Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 2001 98:13090-13095[Abstract/Free Full Text]
  65. Clouthier DE, Avarbock MR, Maika SD, Hammer RE, Brinster RL. Rat spermatogenesis in mouse testis. Nature 1996 381:418-421[CrossRef][Medline]
  66. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes. Biol Reprod 1999 60:515-521[Abstract/Free Full Text]
  67. Dobrinski I, Avarbock MR, Brinster RL. Germ cell transplantation from large domestic animals into mouse testes. Mol Reprod Dev 2000 57:270-279[CrossRef][Medline]
  68. Huang Z, Tamura M, Sakurai T, Chuma S, Saito T, Nakatsuji N. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene. FEBS Lett 2000 487:248-251[CrossRef][Medline]
  69. Ikawa M, Tergaonkar V, Ogura A, Ogonuki N, Inoue K, Verma IM. Restoration of spermatogenesis by lentiviral gene transfer: offspring from infertile mice. Proc Natl Acad Sci U S A 2002 99:7524-7529[Abstract/Free Full Text]
  70. Hecht NB. Molecular mechanisms of male germ cell differentiation. Bioessays 1998 20:555-561[CrossRef][Medline]
  71. Temin HM. Reverse transcription in the eukaryotic genome: retroviruses, pararetroviruses, retrotransposons, and retrotranscripts. Mol Biol Evol 1985 2:455-468[Abstract]
  72. Lower R, Lower J, Kurth R. The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. Proc Natl Acad Sci U S A 1996 93:5177-5184[Abstract/Free Full Text]
  73. Dejucq N, Jegou B. Viruses in the mammalian male genital tract and their effects on the reproductive system. Microbiol Mol Biol Rev 2001 65:208-231[Abstract/Free Full Text]
  74. Paul JH, Jeffrey WH, DeFlaun MF. Dynamics of extracellular DNA in the marine environment. Appl Environ Microbiol 1987 53:170-179[Abstract/Free Full Text]
  75. Graham JB, Istock CA. Genetic exchange in Bacillus subtilis in soil. Mol Gen Genet 1978 166:287-290[Medline]
  76. Stewart GJ, Carlson CA. The biology of natural transformation. Annu Rev Microbiol 1986 40:211-235[CrossRef][Medline]
  77. Cabrera M, Chan PJ, Kalugdan TH, King A. Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues. J Assist Reprod Genet 1997 14:120-124[Medline]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
E. Harel-Markowitz, M. Gurevich, L. S. Shore, A. Katz, Y. Stram, and M. Shemesh
Use of Sperm Plasmid DNA Lipofection Combined with REMI (Restriction Enzyme-Mediated Insertion) for Production of Transgenic Chickens Expressing eGFP (Enhanced Green Fluorescent Protein) or Human Follicle-Stimulating Hormone
Biol Reprod, May 1, 2009; 80(5): 1046 - 1052.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
Y. Yamauchi, B. Doe, A. Ajduk, and M. A Ward
Genomic DNA Damage in Mouse Transgenesis
Biol Reprod, November 1, 2007; 77(5): 803 - 812.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
P. N. Moreira, P. Giraldo, P. Cozar, J. Pozueta, A. Jimenez, L. Montoliu, and A. Gutierrez-Adan
Efficient Generation of Transgenic Mice with Intact Yeast Artificial Chromosomes by Intracytoplasmic Sperm Injection
Biol Reprod, December 1, 2004; 71(6): 1943 - 1947.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
P. R. Tovich, P. Sutovsky, and R. J. Oko
Novel Aspect of Perinuclear Theca Assembly Revealed by Immunolocalization of Non-Nuclear Somatic Histones During Bovine Spermiogenesis
Biol Reprod, October 1, 2004; 71(4): 1182 - 1194.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
68/5/1477    most recent
biolreprod.102.009340v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Celebi, C.
Right arrow Articles by Jégou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Celebi, C.
Right arrow Articles by Jégou, B.
Agricola
Right arrow Articles by Celebi, C.
Right arrow Articles by Jégou, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS