WO2008051620A2 - Methods and compositions for intracytoplasmic sperm injection-mediated transgenesis - Google Patents

Abstract

Methods and compositions for the generation of transgenic animals by intracytoplasmic sperm injection (ICSI) are provided herein. In some embodiments, such methods can include: treating isolated spermatozoa with one of the group consisting of: lysolecithin, digitonin, sonication, and piezo pulses to disrupt or remove the sperm plasma membrane; contacting a sperm thus treated with a nucleic acid mixture that includes a nucleic acid containing a transgene to form a composition; and introducing the composition into an unfertilized oocyte of the same species to form a transgenic embryo. In some embodiments, the nucleic acid mixture includes a nucleic acid containing a transgene flanked by two terminal repeats and one of the group consisting of: a transposase polypeptide and a nucleotide sequence encoding a transposase. In some embodiments, the transposase is a piggyBac transposase.

Description

METHODS AND COMPOSITIONS FOR INTRACYTOPLASMIC SPERM INJECTION-MEDIATED TRANSGENESIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 60/854,310, filed on October 24, 2006, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for the generation of transgenic animals. Particular embodiments relate to the use of plasma membrane-disrupting agents and procedures and modes of use by which they effectively generate transgenic animals when used in in vitro fertilization-coupled transgenesis.

BACKGROUND

[0003] The generation of transgenic animals has great value in both basic and applied genetic research and in commercial applications. Transgenesis relies on the integration of exogenous nucleic acid into a host cell. Integration can be achieved passively, where insertion of a transgene is mediated by host cell DNA repair mechanisms. Transgenesis can also be performed in an active manner, using viruses and viral-based vectors that encode DNA-integrating components. Such methods produce higher frequencies of transgene insertion, but introduce risks associated with the use of attenuated or inactivated viruses and viral vectors. Thus, wider application of transgenic technology will require the development of non-viral transgenesis methods that provide efficient gene integration.

[0004] The process of microinjection of a single spermatozoon directly into an oocyte, commonly known as intracytoplasmic sperm injection (ICSI), has great value in in vitro fertilization in humans and in animals used for commercial or research applications. In ICSI transgenesis, sperm is injected into an oocyte in combination with exogenous DNA encoding a transgene, creating a fertilized, transgenic embryo. For efficient transgenesis, sperm are freeze-thawed immediately prior to ICSI. This breaks down the plasma membrane and enables interaction of the transgenic DNA with the nuclear DNA of the sperm as it decondenses within the oocyte following microinjection. However, the freeze- thawing process is detrimental to embryo development, as it increases the frequency of breakage of the sperm's nuclear DNA. Thus, an increase in the success rate of transgenesis by ICSI will also require development of methods of treating spermatozoa that effectively disrupt or remove their plasma membrane while minimizing damage to their nuclear DNA.

SUMMARY OF THE INVENTION

[0005] Methods and compositions for the generation of transgenic animals by intracytoplasmic sperm injection (ICSI) are provided herein. In some embodiments, such methods can include: treating isolated spermatozoa with one of the group consisting of: lysolecithin, digitonin, sonication, and piezo pulses to disrupt or remove the sperm plasma membrane; contacting a sperm thus treated with a nucleic acid mixture that includes a nucleic acid containing a transgene to form a composition; and introducing the composition into an unfertilized oocyte of the same species to form a transgenic embryo. In some embodiments, the nucleic acid mixture includes a nucleic acid containing a transgene flanked by two terminal repeats and one of the group consisting of: a transposase polypeptide and a nucleotide sequence encoding a transposase. In some embodiments, the transposase is a piggyBac transposase. In some embodiments, the transposase is encoded by a nucleotide sequence on the same nucleic acid containing the transgene. In some embodiments, the nucleic acid encoding the transposase is an mRNA. In some embodiments, the nucleic acid containing a transgene is a linearized plasmid. In some embodiments, the nucleic acid containing a transgene is a bacterial artificial chromosome. [0006] In some embodiments, the plasma membrane of the injected sperm is disrupted or removed by treatment with lysolecithin. In some embodiments, the plasma membrane of the injected sperm is disrupted or removed by treatment with digitonin. In some embodiments, sperm are treated with lysolecithin or digitonin at a concentration, expressed as a percentage by weight, of between 0.001% or less and 0.5% or more. In some embodiments, sperm are treated with lysolecithin or digitonin at a concentration of about 0.02%. In some embodiments, sperm are treated with lysolecithin or digitonin for between 5 seconds or less and 10 minutes or more. In some embodiments, sperm are treated with lysolecithin or digitonin for about 1 minute. In some embodiments, the plasma membrane of the injected sperm is disrupted or removed by treatment with sonication. In some embodiments, treatment with sonication is between 5 seconds or less and 5 minutes or more. In some embodiments, the plasma membrane of the injected sperm is disrupted or removed by treatment with at least one piezo pulse.

[0007] Embodiments of the invention include sperm that have been thus treated to have disrupted or absent plasma membranes. Further embodiments include embryos formed by intracytoplasmic injection into oocytes of sperm thus treated to have disrupted or absent plasma membranes. Yet further embodiments include methods of fertilization of oocytes by sperm thus treated to have disrupted or absent plasma membranes.

Exogenous nucleic acids, sperm, pollen, male gametes, sperm heads, oocytes, ova, female gametes, and the like, obtained from any suitable animal including vertebrates, invertebrates, plants, mammals, fish, amphibians, reptiles, birds, rodents, cats, dogs, cows, pigs, sheep, goats, horses, primates, and the like, are useful in the invention. Embodiments of the present invention include transposable exogenous nucleic acids that are flanked by nucleic acid sequences to form an inverted repeat sequence recognized by a transposase. The exogenous nucleic acid may contain more than one transgene and/or more than one transposable exogenous sequence. Prokaryotic and eukaryotic transposases are useful in the present invention. Embodiments of the invention also encompass chimeric transposases each including a host-specific DNA binding domain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0009] Figure 1 depicts rates of mouse oocyte activation after ICSI- fertilization by sperm subjected to various means of plasma membrane disruption.

[0010] Figure 2 depicts electron micrographs of mouse and human spermatozoa before and after treatment with the plasma-membrane disrupting amphiphilic agent lysolecithin.

[0011] Figure 3 depicts patterns of intracellular Ca²⁺ oscillations within oocytes that signal oocyte activation following microinjection with treated or untreated spermatozoa.

[0012] Figure 4 depicts a microscopic image of mouse spermatozoa following treatment with sonication, which removes their tails and disrupts plasma membrane integrity.

[0013] Figure 5 depicts transgenic mice pups examined for EGFP transgene expression in their skin by epifluorescence following ICSI transgenesis.

[0014] Figure 6 depicts a DNA agarose gel showing PCR analysis of tail samples from newborn mice pups, wherein bands at the positions indicated by arrows reveal the presence of integrated transgenic BAC DNA in individual pups.

[0015] Figure 7 depicts the plasmid designated pMMK-2 containing both the piggyBac transposase gene and a piggyBac transposon, including between its 5¹ and 3' terminal repeats (TRs) the gene for EGFP. The piggyBac transposase gene is driven by the CMV promoter.

[0016] Figure 8 depicts a "transposome" complex formed by a purified transposase similar to piggyBac, the Tn5 transposase, and a Tn5 transposon- donating plasmid.

[0017] Figure 9 depicts Southern blot analysis revealing copy numbers of a transgene in mice generated from Tn5-transposome-injected embryos, as well as an image of an EGFP-transgenic and nontransgenic mouse.

[0018] Figure 10 depicts PCR and Southern blot testing for transposon integration in transgenic mice generated using a Tn5-transposome.

[0019] Figure 11 depicts a diagram of transgenesis using chimeric transposon technology (CTT). A single plasmid encoding a transgene and a chimeric, site-selective transposase, both under the control of an enhancer and promoter, is transfected into a cell. The transposase becomes expressed, binds the terminal repeats flanking the transgene, excises and directs insertion of the transgene into a specific site in the cell's genomic DNA

DETAILED DESCRIPTION

[0020] Embodiments of the invention relate to the discovery that the production of live offspring from oocytes fertilized in vitro by intracytoplasmic sperm injection can be made more efficient by isolating live spermatozoa, subjecting the spermatozoa to a treatment that disrupts or removes their plasma membrane, microinjecting a single plasma-membrane disrupted spermatozoon into an unfertilized oocyte to form a fertilized embryo, and implanting the embryo into a viable mother of the same species. Intracytoplasmic sperm injection (ICSI) has several uses in in vitro fertilization in humans and in animals used for commercial or research applications. Embodiments of the invention provide methods for the production of live offspring by ICSI-mediated in vitro fertilization. Methods of disrupting or removing the plasma membrane from spermatozoa prior to direct injection of spermatozoa into oocytes are comprehended.

[0021] A salient difference between natural and ICSI fertilization is that in the latter, the sperm plasma membrane as well as the acrosome, a sperm organelle containing powerful hydrolyzing enzymes, are introduced into the oocyte. The acrosome caps the anterior end of a spermotozoon head, and the enzymes it contains break down proteins in an exterior protective layer of the oocyte known as the zona pellucida. During normal fertilization, the acrosome membrane and the sperm's plasma membrane fuse as the sperm contacts and traverses the zona pellucida. This releases the enzymes into the zona pellucida to facilitate the sperm's penetration of this protective layer. In ICSI₁ however, whole spermatozoa are injected directly into the oocyte. For species with small acrosomes, injection of the acrosome into an oocyte does not appear to produce serious problems, but for species such as the hamster, cow, and pig, which possess very large acrosomes, injection results in death of the oocyte (Yamauchi, Y. et al, [2002] Biol. Reprod. 67, 534-539). This is believed to be the result of high concentrations of the hydrolyzing enzymes that damage the oocyte interior (Morozumi, K. and Yanagimachi, R. [2005] Proc. Natl. Acad. Sci. USA. 102, 14209-14214).

[0022] Another notable difference between normal and ICSI fertilization is that repetitive transient increases in intracellular Ca²⁺ concentration of the oocyte (Ca²⁺ oscillations), the pivotal signal for oocyte activation (Deguchi, R., et al [2000] Dev Biol 218, 299-313; Miyazaki, S., et al [1993] Dev Biol 158, 62-78; Jones, KT. [1998] I nt J Dev Biol 42, 1-10; and Miyazaki, S. [2006] Semin Cell Dev Biol 17, 233-243), begin much more slowly in ICSI oocytes than in normally fertilized oocytes. In the mouse, for instance, Ca²⁺ oscillations begin one to three minutes after plasma membrane fusion between a fertilizing spermatozoon and an oocyte (Lawrence, Y, Whitaker, M & Swann, K. [1997] Development (Cambridge, UK) 124, 233-241), whereas oscillations begin 15 to 30 minutes after ICSI (Nakano, Y, Shirakawa, H₁ Mitsuhashi, N, Kuwabara, Y & Miyazaki, S. [1997] MoI Hum Reprod 3, 1087-1093; Sato, MS, Yoshitomo, M, Mohri, T & Miyazaki, S. [1999] Cell Calcium 26, 49-58). In human oocytes, Ca²⁺ oscillations start four to 12 hours after ICSI when a plasma membrane-intact spermatozoon is injected (Tesarik, J, Sousa, M & Testart, J. [1994] Hum Reprod 9, 511-518). Ca²⁺ oscillations begin faster (14 ± 6 min) when a spermatozoon is immobilized by applying several piezo pulses to the proximal one-third of the sperm tail before injection. These immobilizing pulses are believed to contribute to the rapid release of sperm factors that initiate oocyte activation (Yanagida, K, Katayose, H, Hirata, S, Yazawa, H, Hayashi, S & Sato, A. [2001] Hum Reprod 16, 148-152). Kasai et al. (Kasai, T, Hoshi, K & Yanagimachi, R. [1999] Zygote 7, 187-193) reported that oocyte activation, assessed by the completion of meiosis, occurred earlier when spermatozoa were freed from the plasma membrane before ICSI. Increased fertilization rates after ICSI using plasma membrane-removed porcine spermatozoa have also been reported (Katayama, M, Sutovsky, P, Yang, BS, Cantley, T, Rieke, A, Farwell, R, Oko, R & Day, BN. [2005] Reproduction 130, 907-916; Tian, JH, Wu, ZH, Liu, L, Cai, Y, Zeng, SM, Zhu, SE, Liu, GS, Li, Y & Wu, CX. [2006] Theriogenology 66, 439^48). Examples of the invention presented herein demonstrate that mouse oocytes developed into live offspring after ICSI regardless of the presence or absence of the sperm plasma membrane, but the proportion of live offspring produced was considerably higher after injection of membrane-free spermatozoa than injection of membrane-intact spermatozoa (Table 1) (Morozumi, K., et al [2006] Proc Natl Acad Sci USA 103, 17661-17666). The referenced study, which is incorporated herein by reference in its entirety, showed that plasma membrane-free spermatozoa activated oocytes earlier than membrane-intact ones (Figures 1 and 3), a result particularly evident for human spermatozoa (Figure ID and Figure 3E-G). It should be noted that human spermatozoa have more stable plasma membranes than the spermatozoa of other species tested (Table 2). If the sperm plasma membrane of a given species is stable and the ability of an oocyte's cytoplasm to "digest" the sperm plasma membrane is low, the membrane will disintegrate slowly or does not disintegrate at all within the oocyte. Quick disintegration of the sperm plasma membrane within the oocyte is important because oocyte activation depends on sperm-borne oocyte-activating factor (SOAF). The strongest candidate for SOAF in mammals thus far is phospholipase C-ζ (Saunders, CM, Larman, MG₁ Parrington, J, Cox, LJ, Royse, J, Blayney, LM, Swann, K & Lai, FA. [2002] Development [Cambridge, L/K/ 129, 3533-3544; Swann, K, Larman, MG, Saunders, CM & Lai, FA. [2004] Reproduction 127, 431^39; Swann, K & Ozil, JP. [1994] lnt Rev Cytol 152, 183-222; Yoda, A, Oda, S₁ Shikano, T, Kouchi, Z, Awaji, T, Shirakawa, H, Kinoshita, K & Miyazaki, S. [2004] Dev Biol 268, 245-257). At least part of SOAF is localized in the perinuclear theca in the postacrosomal region (Kimura, Y₁ Yanagimachi, R₁ Kuretake, S₁ Bortkiewicz, H, Perry, AC & Yanagimachi, H. [1998] Biol Reprod 58, 1407-1415; Fujimoto, S₁ Yoshida, N, Fukui, T₁ Amanai, M₁ Isobe, T₁ Itagaki, C, Izumi, T & Perry, AC. [2004] Dev Biol 274, 370-383; Perry, AC₁ Wakayama, T, Cooke, IM & Yanagimachi, R. [2000] Dev Biol 217, 386-393; Knott, JG, Kurokawa, M & Fissore, RA. [2003] Dev Biol 260, 536-547) and under the plasma membrane over the equatorial segment of the acrosome (Sutovsky, P₁ Manandhar, G, Wu₁ A & Oko, R. [2003] Microsc Res Tech 61, 362-378). When an intact spermatozoon is injected, SOAF will not be exposed to the oocyte's cytoplasm until the sperm plasma membrane in these two regions disintegrates. Embodiments of the invention allow spermatozoa to be freed from plasma membranes before ICSI. Soluble cytosolic SOAF can leak out of the spermatozoon and be lost in the medium following plasma membrane removal. However, the SOAF bound to the perinuclear theca will remain and be exposed to the ooplasm upon injection, resulting in an immediate initiation of Ca²⁺ oscillations. This situation is close to normal fertilization, in which SOAF comes into an immediate contact with the ooplasm upon sperm-oocyte membrane fusion.

[0023] In preferred embodiments of the invention, sperm are demembranated by treatment with the lysophospholipid lysolecithin. Mouse ICSI using lysolecith in-treated spermatozoa resulted in a high percentage of normal live offspring (Table 1). Lysolecithin is a product of hydrolysis of membrane phospholipids by phospholipase A, and is not "alien" to spermatozoa. Lysolecithin plays important roles in various biological processes, including the sperm acrosome reaction (Lessig, J, Glander, HJ, Schiller, J, Petkovic, M, Paasch, U & Arnhold, J. [2006] Andrologia 38, 69-75; Roldan, ER. [1998] Front Biosci 3, D1109-D1119). Although lysolecithin was washed away before ICSI and was not injected into oocytes, a small amount (~1.0 pg) of lysolecithin intentionally injected into each oocyte neither activated oocytes nor affected embryonic and fetal development of fertilized mouse eggs. Other embodiments of the invention encompass the use of Triton X-100 to disrupt or remove the sperm plasma membrane before ICSI. In a preferred embodiment, the plasma membrane disrupting agent is digitonin. Digitonin has enhanced specificity for permeabilizing the plasma membrane, leaving sperm nuclear membranes fully intact.

[0024] Further embodiments of the invention relate to the use of sperm-demembranization treatments for ICSI transgenesis, a procedure in which exogenous plasmid DNA encoding a transgene is co-injected into oocytes along with individual spermatozoa. For efficient transgenesis, sperm are freeze- thawed just prior to ICSI. This treatment is used to destroy the plasma membrane, which enables the interaction of the transgenic DNA with the nuclear DNA of the sperm as it decondenses within the oocyte following microinjection. However, the freeze-thawing process is detrimental to embryo development, as it increases the frequency of breakage of the sperm's nuclear DNA. Embodiments of the invention allow for the removal of the sperm plasma membrane without freeze-thawing of sperm, thereby avoiding the risks of damage to the nuclear DNA. In preferred embodiments, individual sperm are treated with an amphiphilic agent, contacted with exogenous DNA carrying a transgene, and injected into oocytes to form a fertilized transgenic embryo. In some embodiments, the amphiphilc agent is lysolecithin. In other embodiments, the amphiphilic agent is Triton X-100 or digitonin. In yet further embodiments, spermatozoa are treated by sonication prior to ICSI transgenesis. In other embodiments, spermatozoa are treated with at least one piezo pulse or several piezo pulses prior to ICSI transgenesis. [0025] In some embodiments of the invention, sperm is treated with an amphiphilic agent at a concentration, expressed as a percentage by weight, of between 0.001% or less and 0.5% or more prior to ICSI. In some embodiments, sperm is treated with an amphiphilic agent at a concentration between 0.005% and 0.2%. In some embodiments, sperm is treated with an amphiphilic agent at a concentration between 0.02% and 0.1%. In some embodiments, sperm is treated with an amphiphilic agent for between 5 seconds or less and 10 minutes or more. In some embodiments, sperm is treated with an amphiphilic agent for between 10 seconds and 5 minutes. In some embodiments, sperm is treated with an amphiphilic agent for between 30 seconds and 2 minutes. In some embodiments, sperm is treated with an amphiphilic agent for about 1 minute.

[0026] In other embodiments, the plasma-membrane-disrupting treatment is sonication lasting between 5 seconds or less and 5 minutes or more. In some embodiments, the plasma-membrane-disrupting treatment is sonication lasting between 10 seconds and 2 minutes. In some embodiments, the plasma-membrane-disrupting treatment is sonication lasting between 20 seconds and 1 minutes. In other embodiments, the plasma-membrane- disrupting treatment is sonication lasting about 30 seconds, or about 1 minute.

[0027] In yet further embodiments of the invention, the plasma- membrane-disrupting treatment is at least one piezo pulse. In other embodiments, the plasma-membrane disrupting treatment is several piezo pulses.

[0028] Methods of generating transgenic animals via ICSI- transgenesis with plasma-membrane-disrupted sperm have previously been described. See U.S. Provisional Application No. 60/854,310, filed on October 24, 2006, entitled LYSOLECITHIN TREATED SPERM. This application, including all methods, figures, and compositions, is incorporated herein by reference in its entirety.

[0029] Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

[0030] The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1: DEVELOPMENT OF MOUSE OOCYTES INJECTED WITH PLASMA MEMBRANE-INTACT OR PLASMA MEMBRANE-DISRUPTED SPERMATOZOA.

[0031] To prepare mouse oocytes for intra-cytoplasmic sperm injection (ICSI), female B6D2F1 mice, 7-15 weeks of age, were superovulated by intraperitioneal injection of 7.5 international units (IU) of equine chorionic gonadotropin, followed 48 hours later by intraperitioneal injection of 7.5 IU of human chorionic gonadotropin (hCG). Mature oocytes were collected from oviducts 14-16 hours after hCG injection. They were freed from cumulus cells by a 3-minute treatment with 0.1% (wt/vol) bovine testicular hyaluronidase (300 USP units/mg; ICN, Costa Mesa, CA) in Hepes-CZB, a bicarbonate-buffered Chatot, Ziomet, and Bavister medium (CZB) (Chatot, CL, Ziomek, CA, Bavister, BD, Lewis, JL & Torres, I. (1989) J Reprod Fertil 86, 679-688), supplemented with 5.56 mM D-glucose, 20 mM HEPES-Na, pH ~ 7.4, 5 mM NaHCO₃, and 0.1 mg/ml polyvinyl alcohol (cold water-soluble) instead of BSA. The cumulus-free oocytes were thoroughly rinsed and kept in CZB before ICSI for up to 3 hours at 37⁰C. [0032] To prepare spermatozoa for ICSI₁ a drop (~5 μl) of dense sperm mass from a cauda epididymis of the mouse was placed at the bottom of a 1.5-ml centrifuge tube containing 300 μl of CZB for 5-20 minutes at 37⁰C to allow spermatozoa to swim up into the medium. Some spermatozoa that swam into the medium were treated with the amphiphilic agents Triton X-100 (Sigma) (Kimura, Y, Yanagimachi, R, Kuretake, S, Bortkiewicz, H, Perry, AC & Yanagimachi, H. (1998) Biol Reprod 58, 1407-1415) or lysolecithin (LL) (Avanti, Birmingham, AL) by mixing a 500-μl sperm suspension with an equal volume of Hepes-CZB containing 0.04-2.0% (vol/vol) Triton X-100 or 0.04-0.4% (wt/vol) LL. The mixture was vortexed or sonicated for 1 min at O⁰C. Spermatozoa were washed in Hepes-CZB by centrifugation (2,000 x g for 3 minutes) before ICSI. Some spermatozoa were picked up individually and demembranated by treating with 0.02% Triton X-100 or 0.02% LL for 1 minute, washed, and injected into oocytes immediately.

[0033] ICSI was carried out according to Kimura and Yanagimachi (Kimura, Y & Yanagimachi, R. [1995] Biol Reprod 52, 709-720) and Szczygiel and Yanagimachi (Szczygiel, MA & Yanagimachi, R. [2003] in "Intracytoplasmic Sperm Injection," eds. Nagy, A, Gertsenstein, M, Vintersten, K& Behringer, RR. [Cold Spring Harbor Lab Press, Woodbury, NY₁] pp. 1797-1924), with some modifications. An aliquot (25 μl) of sperm suspension was mixed thoroughly with 50 μl of Hepes-CZB containing 8-12% (wt/vol) polyvinylpyrrolidone (M_r 360,000). A drop of this suspension was transferred under paraffin oil (either Squibb & Sons [Princeton, NJ] or Merck [Tokyo, Japan]) in a plastic dish (100 x 100 mm) previously placed on the stage of an inverted microscope equipped with a micromanipulation system. Three types of spermatozoa were injected into oocytes: (/) the entire body of a single, live spermatozoon, (H) a sperm head isolated from the tail by applying a single or a few piezo pulses to the neck region, and (Hi) the head of a single spermatozoon previously treated with either Triton X-100 or LL. Approximately 15 oocytes in a group were operated within 5 minutes. ICSI was completed within 2 hours after collection of oocytes from the oviduct. Following ICSI, the medium used forculturing oocytes was CZB, pH ~7.4, supplemented with 5.56 mM D-glucose and 4 mg/ml BSA, maintained under 5% CO₂ in air (Hepes-CZB used in during oocyte collection and ICSI was used under 100% air). Two-cell embryos developed from ICSI oocytes were transferred into oviducts of pseudopregnant CD1 (albino) females that had been mated during the previous night with vasectomized males of the same strain. Surrogate females were killed on day 19 of pregnancy, and their uteri were examined for the presence of live fetuses. Live fetuses, if any, were collected by Cesarean section and raised by lactating CD1 foster mothers. The difference between the experimental group and matched control group was compared by using Fisher's exact probability test or χ² test. Differences were considered significant at the P < 0.05 level.

[0034] When a single mouse spermatozoon with intact plasma membrane was injected into an oocyte, 80% of ICSI-surviving oocytes developed into blastocysts in vitro. The same proportion of oocytes developed into blastocysts after injection of isolated sperm heads with or without prior treatment of spermatozoa with Triton X-100 or LL (data not shown). However, the method of handling spermatozoa before ICSI affected postimplantation development of embryos (Table 1). When the overall efficiency of embryonic development was assessed by the proportion of full-term fetuses developed from two-cell embryos, the lowest value (40%) was obtained after injection of plasma membrane-intact spermatozoa (experiment A). Developmental efficiency increased (58%) when sperm heads and tails were separated by piezo pulses, and the heads were individually injected into oocytes (experiment B). The highest efficiency (71%) was recorded when spermatozoa were treated individually with 0.02% LL immediately before injection, and their heads were injected individually into oocytes (experiment C). The efficiency was reduced (46%) when spermatozoa were treated with LL as a mass, and LL-treated sperm heads were left in Hepes-CZB medium for 5-60 minutes before ICSI (experiment D). This reduction in developmental efficiency also occurred with sperm heads treated with Triton X-100, as evidenced by the reduced rate of development of normal offspring in experiment F relative to experiment E. Missing upon filing

Table 1. Term development of mouse embryos developed from the oocytes fertilized by injection of intact, immobilized, lysolecithin-treated and Triton X-100-treated mouse spermatozoa

Total no. of No. (%) of zygotes No. of two-cells No. (%) of live

Experiment Sperm Injected zygotes cultured developed to two- transferred (no. of normal offspring

(no. of reps.) cell recipients) at term

A Intact 108 (7) 102 (94) 102 (9) 41 (40.2)^*

B Isolated heads 142 (10) 137 (96) 137 (12) 80 (58.3)*

Lysolecithin-

C treated, 123 (7) 123 (100) 123 (9) 88 (71.5)* individually

Lysolecithin-

D treated, as a 158 (7) 149 (94) 149 (13) 70 (46.9)^s group

Triton X-100-

E treated, 118 (7) 115 (97) 98 (7) 58 (59.2)' individually

Triton X-100-

F treated, as a 120 (9) 118 (98) 118 (11 ) 43 (36.4)¹ group

Experiment (Exp.) A, a motile spermatozoon with intact head and tail was injected into each oocyte; B, only the head was injected after separation of the head and tail by piezo pulses; C and E, spermatozoa were individually treated for 1 min with 0.02% LL or 0.02% Triton X-100 and washed in Hepes-CZB containing 12% PVP for 1 min before injection of a single sperm head isolated from the tail by piezo pulses; D and F, -105 spermatozoa were treated for 1 min with 0.02% LL or 0.02% Triton X-100, washed by centrifugation for 3 min, and left in Hepes-CZB for 5-60 min before injection of a single sperm head isolated from the tail by piezo pulses. * vs. t. P < 0.01 ; t vs. φ, P < 0.01 , t vs. §, P < 0.01 ; U vs. || , P < 0.01.

EXAMPLE 2: TIMING OF OOCYTE ACTIVA TION AFTER ICSI BY USING

SPERMATOZOA WITH OR WITHOUT INTACT PLASMA MEMBRANES

[0035] To assess the timing of oocyte activation following ICSI with spermatozoa with or without intact plasma membranes, the extrusion of the second polar body from the fertilized oocyte was observed as a visible indication of completion of oocyte activation. Activation by mouse, human, porcine, and bovine sperm was compared. Mouse oocyte and spermatozoa isolation was performed as described in Example 1. To prepare human sperm, one to two ml of liquefied human semen was placed at the bottom of a 5-ml tube containing 3 ml of CZB to allow spermatozoa to swim into the medium for 30-60 minutes at 37⁰C. To prepare bovine and porcine sperm, a 500-μl aliquot of bovine or boar semen was placed at the bottom of a 1.5-ml plastic centrifuge tube containing 500 μl of CZB to allow spermatozoa to swim up for 20 min at 37°C. (Frozen bull semen in plastic straws [CRI, Shawano, Wl] was thawed in a 37°C water bath immediately before use, and boar semen stored at 17°C [International Boar Semen, Eldora, IA] was brought to room temperature before use.) Spermatozoa that swam into the medium were either left untreated, immobilized by a single or a few piezo pulses to the neck region (Kuretake, S, Kimura, Y, Hoshi, K & Yanagimachi, R. (1996) Biol Reprod 55, 789-795) to separate the sperm heads from the tail, treated for 1 minute with 0.02-1.0% Triton X-100, or treated for 1 minute with 0.02-0.2% LL. ICSI was then performed as described in Example 1. ICSI oocytes were maintained at 37⁰C, examined every 30 minutes, fixed, and stained as described in Yanagida, K, Yanagimachi, R, Perreault, SD & Kleinfeld, RG. (1991) Biol Reprod 44, 440-447 to verify the completion of the meiotic divisions of oocytes. Because the heads of human spermatozoa, unlike those of spermatozoa of other species, were unable to be separated from the tail by piezo pulses, the entire body of a single spermatozoon was injected regardless of whether it was treated with Triton X-100 or LL. As a rule, only the heads of mouse, bovine, and boar spermatozoa were injected into oocytes.

[0036] Figure λA shows percentages of mouse oocytes activated after ICSI using mouse spermatozoa. When the entire body of an intact, live spermatozoon was injected into each oocyte, few oocytes were activated at 60 minutes after ICSI. It was only after 210 minutes that 100% of injected oocytes were activated. When the sperm head was isolated from the tail by a piezo pulse and injected, oocytes were activated slightly earlier. The earliest oocyte activation took place when spermatozoa were treated with Triton X-100 or LL before injection of isolated sperm heads. Similar results were obtained after injection of bovine and porcine spermatozoa (Figure 1 , B and C). Acceleration of oocyte activation by plasma membrane removal from spermatozoa was most dramatic for human spermatozoa (Figure 1 D). Membrane-intact human spermatozoa often remained motile within the oocyte's cytoplasm for 1 hour after ICSI. Membrane-intact human spermatozoa activated only 20% of mouse oocytes even at 300 minutes after ICSI. More oocytes were activated when spermatozoa were immobilized before injection. The earliest oocyte activation occurred after injection of Triton X-100- or LL-treated- spermatozoa.

[0037] Figure 2 shows electron micrographs of the heads of mouse spermatozoa before (A) and after (S) LL treatment and human spermatozoa before (C) and after (D) LL treatment, (a, acrosome; n, nucleus; p, plasma membrane; pnm, perinuclear material; scale bars, 1 μm.) LL removed both the plasma membrane and the acrosome from spermatozoa (S and D). Note that perinuclear material (theca) remains on the sperm nuclei after removal of the plasma membrane and acrosome.

EXAMPLE 3: ONSET AND PATTERN OF INTRACELLULAR CA²⁺ OSCILLATIONS AFTER ICSI USING PLASMA MEMBRANE-INTACT AND MEMBRANE-DISRUPTED SPERMATOZOA.

[0038] To assess the timing of oocyte activation with injected spermatozoa by a second method, the onset of intracellular Ca²⁺ oscillations in mouse oocytes was determined after injection of (/) motile mouse spermatozoa without any treatments, (H) mouse spermatozoa treated individually with 0.2% LL, (Hi) motile human spermatozoa without any treatments, (/V) human spermatozoa immobilized by applying a few piezo pulses to the midpiece, and (v) human spermatozoa treated individually with 0.2% LL, in each case as described in the previous examples. Repetitive intracellular Ca²⁺ rises are known to trigger oocyte activation (Jones, KT. [1998] lnt J Dev Biol AZ, 1-10; Miyazaki, S. [2006] Semin Cell Dev Biol 17, 233-243; Swann, K & Ozil, JP. [1994] lnt Rev Cytol 152, 183-222). Oocytes were loaded with the Ca²⁺- sensitive fluorescent dye fura-2 acetoxymethyl ester (fura-2 AM; Dojindo Laboratories, Kumamoto, Japan) by incubating them in Hepes-CZB containing 2.5 μM fura-2 AM for 7 minutes at 24°C. After washing, a group of 10-15 eggs was subjected to intracellular calcium ion concentration ([Ca²⁺Ji) measurement by a conventional Ca²⁺ imaging method (Sato, MS, Yoshitomo, M, Mohri, T & Miyazaki, S. [1999] Cell Calcium 26, 49-58) using an image processor (Arugas 200; Hamamatsu Photonics, Hamamatsu, Japan). Three to five oocytes were consecutively injected with spermatozoa at 23-24⁰C and transferred to a dish of M2 medium (Fulton, BP & Whittingham, DG. [1978] Nature 273, 149-151) previously placed on a warmed stage (32-33⁰C) of a UV microscope. Some ICSI was performed directly on the warmed stage of the UV microscope (Figure 3D). Intracellular calcium ion concentration measurement started immediately or within several minutes after onset of ICSI and continued at about 32⁰C because lowering the temperature at this magnitude slows down aging of oocytes and reduces damage by UV illumination without changing the Ca²⁺ response. The time of onset and the pattern of Ca²⁺ oscillations after ICSI are shown in Figure 3. Time 0 represents the moment of ICSI.

[0039] When an intact, motile mouse spermatozoon was injected into an oocyte, the spermatozoon stopped its tail movement mostly within 10 minutes. The first Ca²⁺ transient after normal fertilization (Deguchi, R, Shirakawa, H, Oda, S, Mohri, T & Miyazaki, S. [2000] Dev Biol 218, 299-313) or ICSI (Sato, MS, Yoshitomo, M, Mohri, T & Miyazaki, S. [1999] Cell Calcium 26, 49-58) is characterized by a Ca²⁺ transient peak of longer duration than subsequent Ca²⁺ transients, which superimpose smaller Ca²⁺ oscillations on the larger peak. Using this criteria, the first Ca²⁺ response depicted in Figure 3A and B, was judged to be the first Ca²⁺ transient. Thus, Ca²⁺ oscillations began 7-20 minutes after injection of membrane-intact mouse spermatozoa. Ca²⁺ transients occurred at an interval of 20 minutes. When LL-treated mouse spermatozoa were injected, high-frequency Ca²⁺ oscillations (8-9 spikes in 10 minutes) were recorded in some oocytes (4 of 13 oocytes) from the very beginning of intracellular calcium ion concentration measurement (Figure 3C). Therefore, Ca²⁺ oscillations are assumed to begin immediately after ICSI. The high- frequency Ca²⁺ oscillations continued for ~20 minutes before the interspike interval lengthened to 10 minutes. In 9 of 13 oocytes, the first Ca²⁺ transient displayed a normal pattern, but occurred immediately after ICSI, followed by succeeding Ca²⁺ spikes at an interspike interval of 10-15 minutes (Figure 3D).

[0040] When intact, live human spermatozoa were injected into mouse oocytes, zero out of five oocytes showed Ca²⁺ response during recording for 50- 300 min (Figure 3E). A Ca²⁺ response was observed after injection of piezo- immobilized human spermatozoa, but this response was erratic: when these piezo-immobilized human spermatozoa were injected into seven mouse oocytes, one of seven of these oocytes showed a single Ca²⁺ transient within 50 min of recording (Figure 3F), three oocytes showed one or a few Ca²⁺ oscillations, at 0.5- to 2-hour intervals, during 4 hours of recording, and the other three oocytes showed typical Ca²⁺ oscillations from the beginning of recording. In contrast, high-frequency Ca²⁺ oscillations occurred in each of four oocytes that were injected with a single LL-treated human spermatozoon (Figure 3G). The amount of LL injected into oocytes was negligible because spermatozoa were thoroughly rinsed before injection. LL did not induce any Ca²⁺ increase even when intentionally injected into oocytes (1 pi of 0.2% solution = ~1.0 pg) (data not shown).

EXAMPLE 4: COMPARISON OF THE RESISTANCE OF SPERM PLASMA MEMBRANES TO LYSOLECITHIN AND TRITON X-100 IN VARIOUS SPECIES

[0041] To compare the relative stabilities of the plasma membrane of sperm from various species to treatment with the aphiphilic agents Triton X-100 or lysolecithin, spermatozoa were treated with either 0.04-2.0% (vol/vol) Triton X-100 or 0.04-0.4% (wt/vol) lysolecithin and observed microscopically. Table 2 shows concentrations of Triton X-100 and lysolecithin that immobilize (kill) 100% of spermatozoa within 10 seconds. Although Triton X-100 treatment failed to reveal clear differences in the stability of sperm plasma membranes among several different species, LL treatment clearly indicated that human spermatozoa have the most stable membranes, as judged by resistance to LL, of all the spermatozoa tested.

Table 2. Concentrations of Triton X-100 and LL to immobilize 100% of spermatozoa within 10 seconds (at 24-25⁰C)

Species Triton X-100 (% v/v) Lysolecithin (% wt/vol)

Human 0.01 0.1

Bovine 0.02 0.03

Mouse 0.02 0.02

Porcine 0.015 0.015

Rat 0.015 0.01

Hamster 0.015 0.003

EXAMPLE 5: ICSI TRANSGENESIS WITH SONICATION-TREATED SPERM AND

EGFP TRANSGENE

[0042] To determine the efficiency of ICSI transgenesis using spermatozoa with plasma membranes disrupted and/or removed by sonication, mouse oocytes and spermatozoa isolation was first performed as described in Example 1. Spermatozoa were then subjected to 30 seconds of sonication in a Branson 1510 water sonicator. As shown in Figure 4, this treatment removes the tails from the majority of spermatozoa, leaving the sperm heads shown. Sonicated spermatozoa were combined with a solution containing a linearized plasmid encoding the Enhanced Green Fluorescent Protein (EGFP), with the plasmid at one of three different concentrations indicated below in Table 3. Individual spermatozoa were injected into single oocytes as described in Example 1. Two-cell embryos that developed from ICSI oocytes were transferred into the oviducts of pseudopregnant CD1 (albino) females that had been mated during the previous night with vasectomized males of the same strain. The females were allowed to give birth to their own young, and the newborn pups were examined for EGFP expression in their skin by epifluorescence (Figure 5).

[0043] The efficiency of EGFP transgenic mouse production using this method is shown in Table 3. Using plasmid concentrations of 0.5, 5, and 10 ng/μl, the percentage of transgenic pups out of the total number of oocytes injected (θj, rightmost column in Table 3) was 6.3, 5.0, and 5.0%, respectively. Transgenesis efficiencies observed using non-sonicated sperm are typically slightly lower, in the range of 2.0 to 4.6%.

Table 3. Rates of transgenic mouse generation using ICSI transgenesis with sonicated sperm. and a plasmid carrying an EGFP-encoding transposon.

[0044] Because the removal of the sperm plasma membrane by sonication appears to facilitate more efficient integration of a plasmid-borne transgene, transgenesis was also attempted using a Bacterial Artificial Chromosome (BAC). BACs are able to carry a large region of DNA containing a gene of interest, and they are thus more likely to contain surrounding regulatory sequences necessary for correctly directing the expression of the transgene in the host cell. Spermatozoa sonication and oocyte ICSI was performed as described above, but in this case, sonicated spermatozoa were combined with a solution containing a BAC carrying the chloramphenicol resistance gene instead of an EGFP-encoding plasmid. As described above, two-cell embryos that developed from ICSI oocytes were transferred into the oviducts of pseudopregnant CD1 (albino) females that had been mated during the previous night with vasectomized males of the same strain. The females were allowed to give birth to their own young, and the presence of the transgene in newborn pups was examined by PCR analysis (Figure 6).

[0045] Table 4 below shows that the rate of transgenic pup generation out of the total number of oocytes injected by this method was 4.4%, comparable to the rates of transgenesis observed using the plasmid-borne EGFP transgene.

Table 4. Rate of transgenic mouse generation using ICSI transgenesis with sonicated sperm and a 192 kb Bacterial Artificial Chromosome (BAC) transgene.

EXAMPLE 6: ICSI TRANSGENESIS WITH LYSOLECITHIN-TREATED SPERM AND

EGFP TRANSGENE

[0046] To determine the efficiency of ICSI transgenesis using spermatozoa with plasma membranes disrupted and/or removed by treatment with lysolecithin (LL), mouse oocytes and spermatozoa isolation was first performed as described in Example 1. Spermatozoa were then treated for 1 minute with 0.002-0.2% LL as described in the previous examples. As in example 5, demembranated spermatozoa were combined with a solution containing a linearize plasmid encoding the Enhanced Green Fluorescent Protein (EGFP), with the plasmid at one of four different concentrations indicated below in Table 5. Individual spermatozoa were then injected into single oocytes as described in Example 1. Two-cell embryos that developed from ICSI oocytes were transferred into the oviducts of pseudopregnant CD1 (albino) females that had been mated during the previous night with vasectomized males of the same strain. The females were allowed to give birth to their own young, and the newborn pups were examined for EGFP expression in their skin by epifluorescence (Figure 5).

[0047] The efficiency of transgenic mouse production observed using this method is shown in Table 5. At the lowest concentration of lysolecithin tested, (0.02%), 2.2% of the oocytes injected developed into EGFP transgenic pups, a value within the range normally observed for ICSI transgenesis using untreated sperm. When the concentration of lysolecithin used to demembranate the sperm was increased to 0.02% and to 0.2% (using 5 or 15 ng/μl plasmid), the appearance of transgenic pups increased to 6.7 and 11.4%, respectively. This increase was not solely due to an increased concentration of the plasmid, however, as the efficiency of transgenesis using a plasmid concentration of 15 ng/μl and a lower lysolecithin concentration of 0.02% was only 3.6% transgenic pups of total oocytes injected. Three out of five conditions of lysolecithin treatment and ICSI transgenesis tested resulted in superior embryo development rates, as indicated by the proportion of transgenic animals to animals born being 50% or greater. Thus, removal of sperm plasma membranes by lysolecithin enhances the efficiency of ICSI transgenesis by approximately 2-3 fold, and at several concentrations of transgene-carrying plasmid results in greater embryo development rates.

Table 5. Generation of transgenic mice using ICSI transgenesis with lysolecithin-treated sperm.

EXAMPLE 6: TRANSPOSASE-MEDIATED TRANSGENESIS WITH PLASMA MEMBRANE-DISRUPTED SPERM

[0048] To generate transgenic mice via transposase-mediated transgenesis, ten microliters of 200 nanogram/microliter plasmid pMMK-2, which carries a transposon encoding EGFP flanked by two terminal repeats and the gene for piggyBac transposase (Figure 8) is first mixed with 10 microliters of sperm with plasma membranes disrupted or removed by treatment with lysolecithin, digitonin, sonication, or piezo pulses as described in the previous examples. Each sperm head that has its tail removed in the mixed solution is individually microinjected into a metaphase Il (Mil) arrested matured mouse oocyte as described in Example 1. Two-cell embryos are transferred into the oviducts of pseudopregnant females which are mated with vasectomized males the night before. The females are allowed to give birth to their own young and the newborn pups are examined for EGFP expression in their skin by epifluorescence.

EXAMPLE 7: TRANSPOSASE-MEDIATED TRANSGENESIS USING TRANSPOSASE POLYPEPTIDE COINJECTED INTO MOUSE EMBRYOS WITH TRANSPOSON DONOR PLASMID AND PLASMA-MEMBRANE-DISRUPTED SPERM

[0049] Some embodiments of the present invention relate to methods of generating a transgenic animal or cell using a transposase polypeptide coinjected or cotransfected with a transposon donor plasmid and plasma membrane-disrupted sperm. A transposase such as piggyBac transposase can be used in this manner to generate mice embryos carrying an EGFP transgene. In this prophetic example, a transposase similar to piggyBac, the bacterial Tn5 transposase is described, but the same method can be performed using piggyBac. Delivery and integration of the EGFP-coding transgene into the mouse embryo genome is carried out with the help of a hyperactive mutant of the Tn5 transposase protein designated *Tn5p (Reznikoff WS, MoI Microbiol 47: 1199-1206 (2003); Naumann TA and Reznikoff WS, J Biol Chem 277: 17623- 17629 (2002)) (Figure 8). The *Tn5p:DNA complexes or "transposomes" shown, resembling natural Tn5 transposition intermediates, are formed by allowing the purified transposase to bind to its terminal repeat (ME) recognition sequences in the absence of Mg²⁺ ions. Freshly isolated sperm heads are treated with lysolecithin, digitonin, sonication, or piezo pulses as described in previous examples, co-injected into mouse metaphase Il (MM) oocytes with either naked dsDNA alone or as a ^*Tn5p:DNA complex. The DNA fragment used to construct the transposome contains an EGFP gene driven by a CAG promoter (Ikawa M, et al., FEBS Lett 375: 125-128 [1995]) similar to pMMK-2 (Figure 7). Two-cell embryos are then transferred into oviducts of surrogate females and allowed to develop to full term. All of the resulting Fo transgenic progeny are recognized for transgene expression by epifluorescence of EGFP (Figure 5). Control progeny do not exhibit epifluorescence. Control experiments using ICSI with only transposon DNA produce few or no germline transgenic mice.

Live born pups are screened by PCR for EGFP transgene integration with primers indicated in Figure 10A. The ones found to be positive for the transgene are further challenged for full length transgene insertion (Figure 10B) and their genomic DNA is subjected to Southern blotting to identify the transgene copy number (Figure 10C). Fragments corresponding to perfectly preserved 5' and 3' ends of the transposome can be detected in several animals, indicating the degree of transgene preservation prior to integration, likely due to protection of DNA ends by bound transposase molecules (Figure 10B).

EXAMPLE 8: TRANSPOSASE-MEDIATED TRANSGENESIS USING TRANSPOSASE MRNACOINJECTED INTO MOUSE EMBRYOS WITH TRANSPOSON DONOR PLASMID AND PLASMA-MEMBRANE-DISRUPTED SPERM

[0050] As an alternative to introducing a transposase gene into an oocyte on the same plasmid as the transgene (pMMK-2), the piggyBac transposase gene can also be encoded on an mRNA that is co-introduced into oocytes with a donor plasmid carrying an EGFP transgene, similar to pMMK-2, but lacking the gene for piggyBac transposase. In this case, expression of the transposase is not delayed by transcription of the transposase gene, and genomic integration of the transposon can have a greater chance of occurring before the embryo's first division, thus producing non-mosaic offspring with an integrated copy of the transgene in each of its cells. Capped RNA transcripts are generated in vitro from a plasmid template encoding piggyBac transposase using T3 RNA polymerase (Riboprobe in vitro Transcription System by Promega). This system produces 7-methylguanosine (rτi₇G)-capped RNAs encoding the piggyBac transposase stabilized with 5' and 3' untranslated sequences from the Xenopus laevis β-globin gene. Following transcription, the RNA is treated with DNasel to digest the DNA template. RNA is purified by lithium chloride precipitation, washed twice with 70% ethanol, and resuspended.

[0051] Live sperm are treated with lysolecithin, digitonin, sonication, or piezo pulses as described in previous examples, combined with the RNA and donor plasmid encoding the transgene, and injected into oocytes as described in these examples. Two-cell embryos that develop from ICSI oocytes are transferred into the oviducts of pseudopregnant CD1 (albino) females that have been mated the previous night with vasectomized males of the same strain. The females are allowed to give birth to their own young, and the newborn pups are examined for EGFP expression in their skin by epifluorescence (Figure 5).

EXAMPLE 9: TRANSPOSASE-MEDIATED TRANSGENESIS WITH PLASMA- MEMBRANE DISRUPTED SPERM AND A CHIMERIC TRANSPOSASE WITH A HOST- SPECIFIC DNA-TARGETING DOMAIN

[0052] Directing transgene integration to a unique and safe site on the host chromosome can overcome the hazards of insertional mutagenesis that can result with integrating vectors currently in use. A transposon-based gene delivery system preferably features a custom-engineered transposase with high integration activity and target specificity. Targeting transposon integration to specific DNA sites using chimeric transposases engineered with a DNA binding domain (DBD) has been demonstrated in mosquito embryos containing a plasmid including a unique site recognized by a GAL4 DNA binding domain fused to a transposase (Maragathavally et al., [2006] FASEB J 20, 1880-1882). Such modifications can render a transposase inactive, however, as observed for variants of Sleeping Beauty transposase engineered for target specificity, which have dramatically reduced transposition activity (Wilson et al., [2005] FEBS Lett 579, 6205-6209). The potential for modifications of piggyBac and other transposases has been assessed by testing their activity when fused to a GAL4 DNA binding domain (Figure 11). GALΛ-piggyBac transposase demonstrates transposition activity similar to that of wild-type piggyBac, while GAL4-7o/2 and GAL4-SB11 transposases possessed negligible activity (Wu et al., [2006] PNAS 103, 15008-15013).

[0053] To perform site-specific transgenesis with a chimeric piggyBac transposase and plasma-membrane-disrupted sperm, live sperm are first treated with lysolecithin, digitonin, sonication, or piezo pulses to disrupt or remove their plasma membranes as described in the previous examples. Sperm thus treated are then combined with a nucleic acid encoding both an EGFP transgene flanked by two terminal repeats and the gene for a GAL4-p/ggySac chimeric protein under the control of a constitutively active promoter, and injected into oocytes as described in previous examples. Two-cell embryos that develop from ICSI oocytes are transferred into the oviducts of pseudopregnant CD1 (albino) females that have been mated the previous night with vasectomized males of the same strain. The females are allowed to give birth to their own young, and the newborn pups are examined for EGFP expression in their skin by epifluorescence (Figure 5). EGFP-expressing mice are counted

Claims

CLAIMSWhat is claimed is:

1. A method of generating a transgenic animal comprising: a. isolating live sperm; b. treating said sperm with any one of the group consisting of: lysolecithin, sonication, and piezo pulses to disrupt or remove the sperm plasma membrane; c. contacting said plasma-membrane-disrupted sperm with a nucleic acid mixture comprising a nucleic acid containing a transgene to fqrm a composition; and d. introducing said composition into an unfertilized oocyte to form a transgenic embryo, wherein said transgene is incorporated into the genome of said embryo.

2. The method of claim 1 , wherein said nucleic acid mixture comprises a nucleic acid containing a transgene flanked by two terminal repeats and one of the group consisting of: a transposase polypeptide and a nucleotide sequence encoding a transposase.

3. The method of claim 2, wherein said transposase is a piggyBac transposase.

4. The method of claim 2, wherein said transposase is encoded by a nucleotide sequence on the same nucleic acid containing the transgene.

5. The method of claim 2, wherein the nucleic acid encoding said transposase is an mRNA.

6. The method of claim 1 , wherein the nucleic acid containing a transgene is a linearized plasmid.

7. The method of claim 1 , wherein the nucleic acid containing a transgene is a bacterial artificial chromosome.

8. The method of claim 2, wherein said transposase is a chimeric transposase comprising a host-specific DNA binding domain.

9. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin at a concentration of between 0.001% and 0.5%.

10. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin at a concentration of between 0.005% and 0.2%.

11. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin at a concentration of between 0.02% and 0.1%.

12. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin for between 5 seconds and 10 minutes.

13. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin for between 10 seconds and 5 minutes.

14. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin for between 30 seconds and 2 minutes.

15. The method of claim 1 , wherein said sperm is treated with any one of the group consisting of lysolecithin and digitonin for about 1 minute.

16. The method of claim 1 , wherein said sperm is treated with sonication for a duration of between 5 seconds and 5 minutes.

17. The method of claim 1 , wherein said sperm is treated with sonication for a duration of between 10 seconds and 2 minutes.

18. The method of claim 1 , wherein said sperm is treated with sonication for a duration of between 20 seconds and 1 minute.

19. The method of claim 1 , wherein said sperm is treated with sonication for a duration of about 30 seconds, or about 1 minute.

20. The method of claim 1 , wherein said sperm is treated with at least one piezo pulse.