Outlook Metaphase II transgenesis*

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1 RBMOnline - Vol 4. No Reproductive BioMedicine Online; on web 25 February 2002 Outlook Metaphase II transgenesis* Dr Tony Perry studied the molecular genetics of epididymal sperm maturation at the University of Bristol, UK, from 1989 until taking up an EMBO Fellowship in He is interested in applying molecular biology to problems of mammalian gamete biology and preimplantation embryology. Using the mouse as a model system, Dr Perry is investigating nuclear transfer cloning and sperm injection as means to manipulate mammalian genomes. This work led to the development of metaphase II transgenesis, which he now reviews in this article. Dr Perry is currently Head of the Laboratory of Mammalian Molecular Embryology at the RIKEN Center for Developmental Biology, Kobe, Japan. Dr Tony Perry Anthony CF Perry Advanced Cell Technology, One Innovation Drive, Worcester, Massachusetts, MA 01605, USA Present address: Center for Developmental Biology (CDB), Minatojima-minamimachi, Chuo-ku Kobe, , Japan. Correspondence: Tel: ; Abstract Since its advent in 1974, at least 11 methods have been developed to introduce potentially heritable exogenous DNA (transgenes; tgs) into mammals. These methods are now briefly reviewed in the context of a nascent method that has been demonstrated by microinjection of membrane-depleted sperm heads and tg DNA into metaphase II (mii) oocytes: mii transgenesis. The efficiency of mii transgenesis is at least as high as that of the well-established and prevailing alternative, pronuclear microinjection. Moreover, mii transgenesis promises to facilitate large tg delivery to assist with the generation of disease models and other paradigms in mammalian genome engineering. Keywords: artificial chromosome, metaphase II transgenesis, mouse, oocyte Introduction Transgenesis describes processes by which exogenous (transgene, tg) DNA is caused to become a stable component of a genome (usually gametic or embryonic), potentially heritably. Table 1 summarizes the methods used to produce transgenic mammals. Probably the first mammalian transgenesis was achieved by injecting purified SV40 DNA into the blastocoel cavities of mouse preimplantation embryos (Jaenisch and Mintz, 1974). Adenoviral tgs have subsequently been intravenously injected into females around day 9 of pregnancy (Tsukamoto et al., 1995). However, this method produced offspring in which the tg was gradually lost, did not integrate, and was neither expressed nor transmitted to progeny. Adenoviral vectors can also be used to infect pronuclear mouse zygotes following sub-zonal introduction into the perivitelline space and in one report produced 3/27 (11%) transgenic founders, two of which expressed the tg and all of which transmitted it to offspring (Tsukui et al., 1996). Retroviral genomes have also been investigated as scaffolds for tg vectors. Co-culture of mouse 4- to 8-cell embryos in the presence of Mo-MuLV-infected primary lung fibroblasts resulted in a single founder (of seven offspring) containing the gpt tg used, with no evidence of tg expression reported (Jähner et al., 1985). Transduction of bovine metaphase II (mii) oocytes by a retroviral vector microinjected into the *Paper based on contribution presented at the Alpha meeting in New York, USA, September perivitelline space produced transgenic calves at a high efficiency (4/4 = 100%) (Chan et al., 1998). One of these transmitted the tg via the germ-line to embryos (analysis of the others was not reported), although the tg was not expressed among founders. This lack of expression is not an inherent feature of retroviral tgs, since retroviruses have been used to transduce cultured mouse primordial germ cells (PGC) which can subsequently colonize immature testes following engraftment, to contribute (in 5/13 cases examined) to tgexpressing germ-line transmission (Nagano et al., 2001). This method is restricted by an apparent reliance on aspermatogenic (in this case c-kit-deficient) recipient testes for PGC transfer, and the need for established methods to culture PGC, presently restricted to mice and humans among mammals. Vectors derived from lentiviruses (a class of retrovirus that can infect non-dividing cells) have recently been used to infect rodent pronuclear embryos, resulting in tg-expressing founders in approximately 70% of mouse, and 41% of rat offspring (Lois et al., 2002). The founders transmitted expressed tgs to progeny and the method recapitulated tissue-restricted tg expression in the mouse. A further class of retroviral element, the Mariner family retroviral transposon (retroposon), Sleeping Beauty (SB), has been engineered for heritable transposon mutagenesis in mice (Fischer et al., 2001; Horie et al., 2001). Although not a method of exogenous DNA delivery per se, it promises a way of generating insertional mutants at 279

2 Table 1. Methods of mammalian transgenesis with some of their reported features. Feature Viral methods Non-viral methods SV40 i.v. Ad-pn 4 8C mii PGC Lenti-pn Testis/ pn IVF trans/nt mii fetus infect retro retro retro infect por inject inject Species Mouse Mouse Mouse Mouse Cattle Mouse Mouse, Mouse Various Mouse Cow, Mouse Rat Goat, Pig, Mouse, Sheep Tg size (kb) to >1000 ~5 to 20 to >150 Gene targeting? nd nd nd nd nd nd nd nd 0.2% nd possible nd Micromanipulation? yes no yes no yes no yes/no yes yes no yes yes Relative difficulty low low low low mod high low- mod low high very high low of tg delivery mod % offspring founders a b 80 c 100 ~ Tg copies/integration nd nd >100 nd nd 1 50 Tg expression nd 0 a 67 nd (% founders) % Transmit tg F1 nd ~ ~50 0 nd ~50 References d a The tg persisted episomally without evidence of integration; expression was analysed at 40 days post-birth. b 5/13 c-kit null recipients transmitted the tg following engraftment with transfected PGC. c Data are for mice. d 1. Jaenisch and Mintz, 1974; 2. Tsukamoto et al., 1995; 3. Tsukui et al., 1996; 4. Jähner et al., 1985; 5. Chan et al., 1998; 6. Nagano et al., 2001; 7. Lois et al., 2002; 8. Huang et al., 2000; 9. Gordon et al., 1980, Brinster et al., 1985; 10. Lavitrano et al., 1989, Maione et al., 1998; 11. Cibelli et al., 1998, Wakayama et al., 1999, McCreath et al., 2000, Keefer et al., 2001, Lai et al., 2002; 12. Perry et al., 1999a, linked loci (since intra-chromosomal transposition is favoured; Fischer et al., 2001). In general, however, viral methods of transgenesis are restricted because they (i) necessitate extra cloning steps, (ii) require facilities for recombinant virus propagation and containment, (iii) have a modest insert capacity, and (iv) are potentially prone to recombination events that result in infectious tg-harbouring virions. These general problems help explain why the use of virus-mediated transgenesis is not widespread, even though it is well known (Jähner et al., 1985). Another reason is that transgenesis can be achieved by methods that obviate the need for viruses. In one example (Huang et al., 2000), tg DNA was injected into the immature testes of mice followed by testicular electroporation and selection of spermatozoa expressing the tg marker (mitochondrial yellow fluorescent protein, YFP) for subsequent intracytoplasmic sperm injection (ICSI). This resulted in 100% of transgenic offspring, but the technique is cumbersome and its applicability restricted by the necessity of transfected sperm (precursor) identification, since the transfection efficiency is low. An alternative method utilizes live mouse spermatozoa to deliver tg DNA during IVF (Lavitrano et al., 1989). The method, known as sperm-mediated gene transfer (SMGT), is presumed to work by allowing an association between sperm membrane components and exogenous (tg) DNA, followed by internalization of the tg (Lavitrano et al., 1992). When tg integration subsequently occurs, it apparently does so at a restricted number of loci, possibly one (Lavitrano et al., 1989; Maione et al., 1998). Moreover, critical parameters governing the prescriptive reproducibility of the method have not been described (Brinster et al., 1989) and it has not been widely adopted outside the laboratory from which it originated. Instead, reproducible transgenesis has been effected by three strategies involving micromanipulation of zygotes or mii oocytes. One entails nuclear transfer (nt) from cultured donor cells that have been transfected in vitro. Cloned offspring generated in this way harbour one or more mutations previously characterized in the cells from which they are cloned prior to cloning. This makes the method potentially powerful because it means that cells (i.e. genomes) with the desired mutation can first be selected before animals are subsequently cloned from them by nt. This two-step approach has been applied to generate transgenic sheep (Schnieke et al., 1997), cattle (Cibelli et al., 1998), mice (Wakayama et al., 1999), goats (Keefer et al., 2001) and pigs (Lai et al., 2002) and can more specifically be used to generate offspring harbouring targeted mutations, at least for mice (Wakayama et al., 1999), sheep (McCreath et al., 2000) and pigs (Lai et al., 2002). Although this method holds great promise, its application is currently confounded by the high degree of technical difficulty that must be overcome to achieve it, and by phenotypes putatively associated with cloning by nt, which are as yet poorly characterized (Wakayama et al., 1999; Tamashiro et al., 2000; Ogonuki et al., 2002). The prevailing method of transgenesis in the 1990s involved microinjecting tg DNA into the pronucleus (usually the male

3 pronucleus, which is the larger of the two) of a 1-cell embryo (Gordon et al., 1980). In the mouse, some 10 20% (sometimes more) of the total number of offspring are transgenic (Gordon and Ruddle, 1981; Brinster et al., 1985) and the method can deliver large tgs in the megabase range (Schedl et al., 1993; Co et al., 2000). However, transgenesis by pronuclear microinjection has drawbacks. Microinjection of large tgs via the small needle tips required (1 2 µm diameter) is confounded by the high viscosity of their solutions, which makes handling difficult. Moreover, shear forces at such fine apertures predispose to chromosome breakage (discussed below). Although pronuclear microinjection can be used to generate transgenic animals of other species including rats, cattle, sheep and pigs (Hammer et al., 1986), it does so with a markedly reduced efficiency (e.g. <2% for cattle) compared with the rate obtained in mice (Wall et al., 1992; Wall, 1997). Table 2. Embryo development and mii transgenesis in vitro. Transgene c Sperm head No. oocytes treatment d Survived m/b % e Total % positive f egfpuntreated (83) a 34 (26) egfptx (79) a 137 (64) egfpft (50) b 127 (82) egfpfd (55) b 134 (87) LacZ FT (73) a 103 (94) LacZ FD (78) a 98 (92) a,b Values with different superscripts within the same column differ significantly (P < 0.05). c Exogenous, tg DNA was at 5 10 µg/ml. The egfptg used throughout the experiments reported in Tables 2 5 is encoded by the 3.5 kb Sal GI-Bam HI fragment of pcx-egfp(perry et al., 1999a). The LacZ reporter is encoded by linearized px-canlacz (Perry et al., 1999a). d Untreated, fresh; TX, TX-100; FT, freeze-thaw; FD, freeze-dry. Details of sperm treatments are described in Perry et al., 1999a. e Development to the morula/blastocyst (m/b) stage at embryonic day 3.5 (E.3.5). Percentages are of oocytes surviving. f The total percentage of m/b containing positive blastomeres including -ve/+ve mosaics and mosaics whose blastomeres all exhibited tg expression. Table 3. Effect of sperm washing following egfptg DNA mixing on the proportion of subsequently fluorescent blastocysts. Sperm head treatment b No. oocytes Survived m/b (%) c Positive (%) d Washed (75) a 71 (62) a Not washed (71) a 66 (80) a a Values within each column do not significantly differ. b Heads were mixed with the egfptg fragment and either washed briefly or not washed in parallel prior to microinjection (Perry et al., 1999a). All three membrane disruption procedures are represented. c Percentages are of surviving oocytes developing to the morula/blastocyst (m/b) stage at E.3.5. d E.3.5 fluorescent m/b including +ve/ ve mosaics and those whose blastomeres all exhibited tg expression. The method has additional technical barriers in larger species; relative to mouse pronuclear zygotes, those of many species are opaque (making a pronucleus undiscernable and difficult to inject with precision), expensive and difficult to obtain. The remainder of this article will describe an emergent method of transgenesis which is straightforward, efficient, and which has the clear potential to circumvent several of the problems associated with its antecedents (Perry et al., 1999a). The method, known as mii transgenesis, entails coinjection of a nucleus-tg DNA mixture into an unfertilized, metaphase II (mii) oocyte. It has been demonstrated in the mouse by intracytoplasmic sperm injection (ICSI); in other words, by microinjecting a sperm head (nucleus) plus tg DNA into an unfertilized oocyte. Efficient tg delivery by mii transgenesis Transgenesis (mii transgenesis) can be achieved by mixing with spermatozoa a tg construct that drives the ubiquitous (e.g. early embryonic) expression of enhanced green fluorescent protein (egfp), and coinjecting them into an (unfertilized) mii oocyte (Perry et al., 1999a). If untreated motile spermatozoa and the egpf tg are coinjected, approximately 1 in 4 of the ensuing blastocysts contain fluorescing (i.e. transgenic) blastomeres after 3.5 day culture (E.3.5) (Table 2). In this case, sperm membranes were intact moments before ICSI, but became broken during microsurgical dissection of the sperm head just prior to injection. Sperm heads can support full development in ICSI even when considered dead in that they are membrane-depleted (Wakayama et al., 1998; Perry et al., 1999a). Sperm membrane disruption methods that permit full development following ICSI include freeze-thawing (Wakayama et al., 1998), freeze-drying (Wakayama and Yanagimachi, 1998), or exposure to detergents such as Triton X-100 (TX-100; Perry et al., 1999b). Mixture of spermatozoa that have been membrane-challenged (as determined by electron microscopy) with tg DNA prior to microinjection results in a significantly higher proportion of fluorescing E.3.5 embryos, ranging from 64% (TX-100) to 87% (freeze-dry), compared with controls using untreated spermatozoa (Table 2). Membrane disruption procedures can thus enhance mii transgenesis (Perry et al., 1999a). However, it was noted that development was attenuated in freeze thaw and freeze-dry groups at rates significantly higher than those of controls (Table 2). To test whether this high rate of developmental failure was linked to the high rate of transgenesis, similar experiments were performed using an analogous tg containing a lacz reporter. Coinjection of mii oocytes with lacz reporter DNA fragments and either freezethawed or freeze-dried spermatozoa generated a high proportion (92 94%) of embryos expressing the lacz tg product, β-galactosidase, without depressing development (Table 2). This shows that high rates of transgenesis do not of themselves abrogate development to the blastocyst stage. It is possible that the rates of transgenesis in Table 2 do not reflect tg integration, but merely episomal persistence of tg DNA and/or associated gene products in embryos. This objection is valid, and evidence suggesting that tg expression means tg integration is discussed below. Assuming it does 281

4 282 Table 4. Injection of egfptg DNA in the absence of an exogenous nucleus (sperm head) fails to produce fluorescing embryos. Material injected and No. oocytes order of injection a Survived m/b (%) b Positive b 1 Sperm head 2 egfptg (79) 0 1 egfptg 2 Sperm head (69) 0 egfptg alone c (98) 0 a Sperm head preparation was by the freeze thaw method (Perry et al., 1999a) Serial injection was separated by min. Linear egfptg DNA (see footnote to Table 2) was at 7 µg/ml. b Percentages are of embryos that had developed to the morula/blastocyst (m/b) stage on E.3.5. Of these, the number with at least one fluorescing blastomere were scored positive (+ve). c Parthenogenetic activation pursuant to tg injection was by SrCl2 in the presence of cytochalasin B. mean this, then the data presented in Table 2 show that membrane disruption greatly favours mii transgenesis and imply that the sperm membrane is a significant barrier to an association between tg DNA and submembrane structures, perhaps including the physiologically basic (positively charged) perinuclear matrix. Supporting this notion, spermatozoa that had been washed with fresh media after being mixed with egfptg DNA retained the ability to produce fluorescent blastocysts, albeit with a slightly reduced efficiency (62% versus 80%) compared with their non-washed counterparts (Table 3). This lack of a significant drop in the efficiency of transgenesis suggests that prior to microinjection, a sperm-tg DNA interaction occurs that is above the threshold of detection. It was investigated whether the interaction might nevertheless occur following microinjection (i.e. inside the egg) in some cases. The answer is (usually) no; not, at least, under the conditions of these experiments. If membrane-disrupted sperm heads and egfptg DNA are microinjected serially (in either order) with no mixing prior to injection, the resulting blasocysts consistently fail to fluoresce (Table 4). Not only that, but injection of the egfptg DNA alone followed by parthenogenic activation permits good parthenogenetic development in which few, if any, of the resulting blastocysts exhibit observable tg expression (Table 4). These data not only argue that an association between spermatozoa and tg DNA before microinjection greatly favours transgenesis, but give us a strong clue that absent an origin of replication, tg DNA does not persist embryonically in an episomal form; it must be integrated, presumably into the paternal genome. Thus, in some cases at least, the observation of tg expression in blastocysts is a good indication of tg integration, since they are apparently causally linked. Table 5. Development to term and tg expression in offspring generated via mii transgenesis protocols. Sperm head Oocytes treatment b Survived m/b Total pups Positive transferred c (%) d (green) pups (%) e Freeze-dry (4) 14 (21) 3 (21) a Freeze thaw (3) 12 (23) 2 (17) a Triton X (9) 31 (21) 6 (19) a a Values do not significantly differ. b Details of sperm treatments are described in Perry et al., 1999a. c m/b, morulae/blastocysts, with the number of surrogate mothers used as recipients in embryo transfers italicized in parentheses. d Percentages (parentheses) are of embryos that were transferred giving rise to live offspring. e Percentages (parentheses) are of the total number of neonatal pups delivered for each sperm treatment that appeared to fluoresce green under long wavelength (480 nm) ultraviolet light. There is additional evidence in support of this causal link, and it comes from showing that the egfptransgenic embryos can develop into transgenic offspring (Table 5). In fact, a high proportion (17 21%) of offspring were transgenic with respect to observable ectopic egfpexpression independent of the membrane disruption method used to prepare spermatozoa prior to microinjection (Table 5) (Perry et al., 1999a). Genotypic analysis by Southern blotting and/or PCR showed that all fluorescing founders indeed contained one or more genomic tg copies, with copy numbers in the range 1 to >50; in these experiments, 73% of founders tested transmitted an expressed tg to offspring (Perry et al., 1999a). The observed frequencies and patterns of mii transgenesis are reminiscent of those achieved by efficient transgenic core facilities using pronuclear microinjection. Yet mii transgenesis is a novel method. The efficiency of transgenesis by pronuclear microinjection was approximately 5% at its inception (Gordon et al., 1980; Brinster et al., 1981) and has since become approximately 3-fold more efficient in mice; a similar improvement in the efficiency of mii transgenesis would routinely produce rates of approximately 60%. Improving modifications of mii transgenesis could include alternative methods of demembranation, to allow greater access of tg DNA to sub-membrane compartments. Enhanced tg access to key nuclear components, possibly including the paternal genome, could be effected by reagents that loosen the nucleus by causing it to swell (e.g. Na + ) and/or by reducing disulphide bonds (e.g. reducing agents such as dithiothreitol and reduced glutathione). Application to different species By obviating the need for pronuclear zygotes, mii transgenesis holds the promise of marked improvements in transgenic livestock production. Transgenesis by pronuclear microinjection, the favoured method to date, is much less efficient in large species than it is in mice; about 1% of offspring are transgenic (Wall et al., 1992; Wall, 1997). This is

5 Table 6. Embryo development in mii transgenesis with large tg constructs. Construct a Construct Sperm No. Development in vitro c No. No. live No. (%) Germ line size (kb) treatment b surviving transferred offspring offspring transmission oocytes embryos transgenic (%) e 8C m b (recipients) d TV FT (12) 14 (8) 6 (43) 5/5 (100) 4 TV TX (15) 63 (31) 14 (21) 12/13 (92) BAC 131 FT/TX (4) 10 (11) 2 (20) 2/2 (100) BAC 170 TX (4) 9 (17) 1 (11) 1/1 (100) BAC 45 TX 14 (1) 12 (86) 5 (42) 4/4 (100) MAC 79.5, 89 FT (7) 19 (14) 9 (47) nd 9 MAC 79.5, 89 TX (10) 83 (47) 13 (16) nd Total (%) (58) 255 (27) 61 (24) 24/25 (96) a TV, targeting vector; BAC, bacterial artificial chromosome; MAC, mammalian artificial chromosome construct. Details are given in Perry et al., b FT, freeze thaw; TX, Triton X-100 extraction. Details are given in Perry et al., c After culture in vitro for 3.5 days (E.3.5). b, blastocyst (including embryos in which the nascent blastocoel cavity is still expanding); m, morula; 8C, noncompacted embryo of at most 8 cells. d Includes transfer of 1- and 2-cell embryos after 1 2 days of culture in vitro; these embryos are therefore not included in preceding columns. e Germ line transmission was assessed for most, but not all, founders. partly because unlike mouse zygotes, which are typically translucent, those of many other species are opaque, so that it is not possible to locate their pronuclei without first subjecting the embryos to potentially traumatic procedures such as centrifugation. Since mii transgenesis does not require the pinpoint accuracy required to locate and deliver DNA into a pronucleus, this trauma is unnecessary. Indeed, pronuclear zygotes themselves are unnecessary; although they are produced routinely in the mouse, their production in larger species is far less trivial. Together, these factors make transgenesis by pronuclear microinjection inefficient and expensive; a single transgenic livestock animal costs in excess of US$100,000 (sometimes much more) to produce. The advent of mii transgenesis promises a reduction in animal usage; even a modest improvement in efficiency would represent a substantial saving per capita of transgenic animals produced. Large tg delivery via mii transgenesis However, mii transgenesis already shows promise in facilitating the increasingly desirable delivery of large constructs (Perry et al., 2001). This has been demonstrated in experiments with larger tgs (of >10 kb) corresponding to tightly spatiotemporally regulated transcripts, and with bacterial and mammalian artificial chromosome constructs (BACs and MACs) (Table 6). Coinjection of larger tg constructs with spermatozoa treated either by freeze-thawing or extraction with TX-100 yielded offspring that were transgenic in 11-47% of cases; Southern hybridization analysis suggested that integration patterns were quasi-random in a manner akin to those observed after transgenesis by pronuclear microinjection. Founders exhibited tg expression characteristic of their corresponding endogenous genes, and transmitted their tgs to offspring in 96% of cases (Table 6). Since the constructs in this study ranged up to around 170 kb, larger tg sizes are apparently not a barrier to efficient mii transgenesis. Moreover, transgenesis was successful using these larger constructs at tg concentrations equivalent to an average of 15 injected molecules. It is therefore possible that constructs are concentrated on, and stabilized by, interactions with the sperm head prior to microinjection, as if the sperm head becomes a Trojan horse to assist tg entry into the oocyte. Association of tgs with sperm head supramolecular structures could afford protection by minimizing the effects of shear forces at the microinjection pipette tip. In addition, such forces are at any rate reduced in comparison to those present at tips used for pronuclear microinjection, in which the smaller aperture generates high pressures; a tip diameter of 1 µm used for pronuclear microinjection corresponds to 0.78 µm 2, compared with up to around 10 µm (78 µm 2 ) for mii transgenesis. The increasing need for large constructs in transgenesis comes in part from their ability to buffer gene expression from position effects so that they are more likely to be faithfully expressed (Giraldo and Montoliu, 2001). It will also in many cases obviate the need to sub-clone genes of interest from clones obtained from artificial chromosome libraries. The efficacy of mii transgenesis with large tg cargoes therefore introduces new opportunities for controlled expression in the dissection of genome function. This need will increasingly be fuelled by the study of polygenic diseases such as hypertension, epilepsy, stroke, obesity, Crohn s disease, lupus, multiple sclerosis and certain cancers such as those of the prostate, breast and colon. The study of such multigenic interactions will be facilitated by an enhanced ability to introduce larger genes and gene clusters in the megabase range; mii transgenesis promises to achieve this. 283

6 284 References Brinster RL, Chen HY, Trumbauer M et al Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, Brinster RL, Chen HY, Trumbauer ME et al Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proceedings of the National Academy of Sciences of the USA 82, Brinster RL, Sandgren EP, Behringer RR, Palmiter RD 1989 No simple solution for making transgenic mice. Cell 59, Chan AWS, Homan EJ, Ballou LU et al Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proceedings of the National Academy of Sciences of the USA 95, Cibelli JB, Stice SL, Golueke PJ et al Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells. Nature Biotechnology 16, Co DO, Borowski AH, Leung JD et al Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection. Chromosome Research 8, Fischer SE, Wienholds E, Plasterk RH 2001 Regulated transposition of a fish transposon in the mouse germ line. Proceedings of the National Academy of Sciences of the USA 98, Giraldo P, Montoliu L 2001 Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Research 10, Gordon JW, Ruddle FH 1981 Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214, Gordon JW, Scangos GA, Plotkin DJ et al Genetic transformation of mouse embryos by microinjection of purified DNA. Proceedings of the National Academy of Sciences of the USA 77, Hammer RE, Pursel VG, Rexroad CE Jr et al Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315, Horie K, Kuroiwa A, Ikawa M et al Efficient chromosomal transposition of a Tc1/mariner-like transposon Sleeping Beauty in mice. Proceedings of the National Academy of Sciences of the USA 98, Huang Z, Tamura M, Sakurai T et al In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene. FEBS Letters 487, Jaenisch R, Mintz B 1974 Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proceedings of the National Academy of Sciences of the USA 71, Jähner D, Haase K, Mulligan R, Jaenisch R 1985 Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection. Proceedings of the National Academy of Sciences of the USA 82, Keefer CL, Baldassarre H, Keyston R et al Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitromatured oocytes. Biology of Reproduction 64, Lai L, Kolber-Simonds D, Park K-W et al Production of α- 1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 298, Lavitrano M, Camaioni A, Fazio VM et al Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57, Lavitrano M, French D, Zani M et al The interaction between exogenous DNA and sperm cells. Molecular Reproduction and Development 31, Lois C, Hong EJ, Pease S et al Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, Maione B, Lavitrano M, Spadafora C, Kiessling AA 1998 Spermmediated gene transfer in mice. Molecular Reproduction and Development 50, McCreath KJ, Howcroft J, Campbell KH et al Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405, Nagano M, Brinster CJ, Orwig KE et al Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proceedings of the National Academy of Sciences of the USA 98, Ogonuki N, Inoue K, Yamamoto Y et al Nature Genetics 30, in press. Perry ACF, Wakayama T, Kishikawa H et al. 1999a Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, Perry ACF, Wakayama T, Yanagimachi R 1999b A novel transcomplementation assay suggests full mammalian oocyte activation is coordinately initiated by multiple, submembrane sperm compartments. Biology of Reproduction 60, Perry ACF, Rothman A, de las Heras JI et al Efficient metaphase II transgenesis with different transgene archetypes. Nature Biotechnology 19, Schedl A, Montoliu L, Kelsey G, Schütz G 1993 A yeast artificial chromosome covering the tyrosinase gene confers copy numberdependent expression in transgenic mice. Nature 362, Schnieke AE, Kind AJ, Ritchie WA et al Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278, Tamashiro KLK, Wakayama T, Blanchard RJ et al Postnatal growth and behavioral development of mice cloned from adult cumulus cells. Biology of Reproduction 63, Tsukamoto M, Ochiya T, Yoshida S et al Gene transfer and expression in progeny after intravenous DNA injection into pregnant mice. Nature Genetics 9, Tsukui T, Kanegae Y, Saito I, Toyoda Y 1996 Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs. Nature Biotechnology 14, Wakayama T, Yanagimachi R 1998 Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nature Biotechnology 16, Wakayama T, Whittingham DG, Yanagimachi R 1998 Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection. Journal of Fertility and Reproduction 112, Wakayama T, Rodriguez I, Perry ACF et al Mice cloned from embryonic stem cells. Proceedings of the National Academy of Sciences of the USA 96, Wall RJ 1997 A new lease on life for transgenic livestock. Nature Biotechnology 15, Wall RJ, Hawk HW, Nel N 1992 Making transgenic livestock: genetic engineering on a large scale. Journal of Cell Biochemistry 49,

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