Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O

Transgenic (Tg) pigs with organ/tissue-specific fluorescence expression provide invaluable research tools for many types of studies, such as organogenesis analysis, in vitro tissue generation from pluripotent cells, and progenitor/stem cell transplantation therapy. We aimed to develop a Tg pig characterised by pancreas- and liver-specific fluorescence expression. A 8.4 kb transgene construct expressing Venus (green fluorescence) under the control of the mouse Pdx1 (pancreatic duodenal homeobox-1) promoter and a BAC-derived construct (170 kb) consisting of the whole-length porcine albumin (Alb) promoter and humanized Kusabira-Orange (huKO, red fluorescence) was introduced into porcine in vitro-matured oocytes using the intracytoplasmic sperm injection (ICSI)-mediated gene transfer method. Injected embryos were transferred to the oviducts of oestrus-synchronized recipients after culture for 1 to 3 days. The transfer of 370 Pdx1-Venus embryos into 4 recipients produce 22 (5.9%) fetuses/piglets, and 9 (40.9%) Tg pigs exhibited pancreas-specific Venus expression. Two (1 male and 1 female) founder Pdx1-Venus-Tg pigs were mated with wild-type (WT) pigs and produced 32 offspring in 3 litters, of which 16 (50.0%) were transgenic. Pancreas-specific Venus expression was inherited in these Tg offspring. The transfer of 523 Alb-huKO embryos into 4 recipients resulted in 19 (3.6%) piglets including a Tg female, which showed liver-specific huKO fluorescent expression. Expression of huKO was detected by RT-PCR exclusively in liver, but not in 7 other organs/tissues examined, including heart, lung, stomach, small intestine, spleen, kidney and skin. This founder Tg female produced a total of 12 non-Tg and 5 Tg offspring (in 2 litters) after mating with a WT boar. Liver-specific huKO expression was inherited in these Tg offspring. Furthermore, the mating of a female Pdx1-Venus pig with an Alb-huKO boar yielded 7 non-Tg and 10 Tg pigs. Four of these Tg pigs carrying both of the transgenes exhibited both pancreas-specific Venus and liver-specific huKO expression in single individuals. Double-Tg pigs with pancreas-specific green fluorescence and liver-specific red fluorescence grew normally, and tests of their reproduction ability are currently underway. These data demonstrate that transgene introduction by ICSI-mediated gene transfer into in vitro-matured oocytes is a feasible option for generating pigs expressing fluorescent proteins in a tissue-specific manner.

Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI-MGT yielded significantly more embryos with non-mosaic transgene expression (P < 0.01). In the ICSI-MGT group, 61.5% (24/39) of the embryos were EGFP-positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP-positive in the PN-injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI-MGT was transgenic, suggesting that ICSI-MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI-MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple-copy, single-site integration of a transgene is the primary outcome of ICSI-MGT in the pig and that ICSI-MGT is less likely than PN injection to cause transgene integration in a mosaic manner.

High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation

ICSI-mediated gene transfer is a powerful technique used to produce transgenic mice and pigs. However, this method of transgenesis has not been applied in bovine due to low embryo development, which is presumed to be a consequence of a failure in sperm factor delivery after ICSI in this species. To bypass this problem, we assisted ICSI with chemical activation, employing two Ionomycin (Io) exposures and 6-Dimethylaminopurine (DMAP) or a novel drug, Dehydroleucodine (DhL). Cumulus–oocyte complexes were aspirated from ovaries obtained from a local slaughterhouse and in vitro matured in bicarbonate-buffered TCM-199 containing 10% FBS, 10 μg mL–1 FSH, 0.3 mm sodium pyruvate, 100 μm cysteamine and 10 UI mL–1 penicillin. IVM conditions were 6% CO2 in humidified air at 39°C for 24 h. MII oocytes were selected and used immediately for ICSI. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construction (pCX-EGFP) was carried out in Na citrate 2.8%, with 0.5 μg plasmid million–1 spermatozoa for 5 min at 0°C. Then, spermatozoa were used for ICSI. Injected oocytes were activated in 5 μm Io for 4 min and placed in TCM-199 for 3 h to allow second polar body emission. Afterwards, some of the oocytes were subjected to a second exposure of Io. Oocytes exposed once or twice to Io were then incubated with 2 mm DMAP (groups Io-DMAP and 2Io-DMAP) or 5 mm DhL (groups Io-DhL and 2Io-DhL) for 3 h. Control groups (Io and 2Io) were not treated with DMAP or DhL. Embryos were cultured in the IVM droplets. EGFP expression was daily evaluated in fluorescence microscope under blue light (488 nm). Significant differences between groups were evaluated by Fisher test (Table 1). DhL chemical activation improved neither development nor transgenesis rates. The double Io exposure significantly improved embryo development. The second exposure to Io previous chemical activation with DMAP resulted in an increase in the percentage of EGFP-expressing embryos. Our results indicate that activation with double Io-DMAP could be considered an alternative assistance for ICSI mediated gene transfer in bovine. Table 1.Effect of activation assisting transgenic ICSI on development and expression of bovine embryos

Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method

Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.

糖尿病モデルトランスジェニッククローンブタの作出 I. ICSI-mediated Gene Transfer(ICSI法) と体細胞核移植の組合わせによるトランスジェニック・クローンブタの効率的作出

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has been described as a technique to obtain transgenic offspring in mice. However, this approach has had limited success in domestic animals due to poor embryo development after ICSI. A first experiment was designed to improve embryo development comparing ICSI-mediated gene transfer with or without chemical activation (CA) in the ovine species. In the second experiment, ICSI-mediated gene transfer assisted by CA was used in porcine, feline, equine, and bovine species. Maturation and culture were done by standard procedures. Semen was collected by artificial vagina in ovine and bovine species. In pigs, ejaculates were obtained using the gloved-hand method, and in feline and equine species, sperm were obtained from epididymides. Samples were frozen by standard means. Thawed spermatozoa were washed twice in Na citrate at 2.8% with 100 µm EDTA at 495g for 5 min and resuspended in Na citrate with 0.5 µg of pCX-EGFP/million spermatozoa for 5 min at 0�C. The pCX-EGFP plasmid contained the egfp gene expressed under chimerical CMV-IE-chicken β-actin promoter control. Sperm cells were immediately injected into the metaphase II oocyte and CA was induced by incubation in TALP-HEPES with 5 µm ionomycin for 4 min, cultured in TCM199 for 3 h, and transferred to a droplet of 1.9 mm 6-dimethylaminopurine (DMAP) for 3 h. During the in vitro culture, exposure to blue light (488 nm) was performed to determine the percentage of green embryos, mosaic expression, and earliest stage of egfp expression. Fluorescence in situ hybridization analysis was performed labeling pCX-EGFP plasmid by nick translation for use as a probe. Statistical analysis was done by chi square. In ovine species, development to blastocyst stage (0/88 v. 3/86; P &gt; 0.05) and number of green embryos (24/88 v. 39/86; P &lt; 0.05) were greater with CA. The egfp expression in ovine embryos assisted by CA began at the 2- (7/39), 4- (9/39), or 8-cell (23/39). However, the expression in ovine embryos without CA occurred only at the 8-cell stage (24/24) stage. In porcine, bovine, feline, and equine species, green embryos were detected at a high proportion (33/55, 10/44, 9/35, and 5/17, respectively), and the percentage of fluorescent blastocysts was 2.3, 2.9, and 9.1% for ovine, feline, and bovine species, respectively. The egfp expression in porcine and feline embryos started at the 2-cell stage (36 and 22%, respectively), whereas it began in bovine and equine embryos at the 4-cell stage (9 and 40% respectively). All species showed a high frequency of mosaic expression (range 60-85%), and the preliminary FISH analysis demonstrated a variable number of integration events in porcine embryos. To our knowledge, this is the first report of exogenous DNA expression in feline and equine embryos. These results suggest that the CA accelerates and increases the pCX-EGFP expression in ovine embryos in agreement with previous studies that have shown earlier expression of genes for parthenogenetic and cloning embryos, both assisted by CA. In conclusion, ICSI-mediated gene transfer assisted by CA can be used to obtain exogenous gene-expressing embryos in domestic species with potential scientific and commercial interests.

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

ブタICSI-mediated gene transferにおける外来遺伝子組み込み様式

We previously reported that transgenic (TG) pigs can be produced from in vitro-matured oocytes using intracytoplasmic sperm injection-mediated gene transfer (ICSI-mediated method) (Kurome et al. 2006 Transgenic Res. 15, 229–240). We subsequently studied the expression of a foreign gene which had been introduced by the ICSI-mediated method. We found that the ICSI-mediated method is considerably less likely than the pronuclear microinjection method to produce embryos in which transgene-positive and transgene-negative cells co-exist, that is, mosaic embryos (Saito et al. 2006 Reprod. Fertil. Dev. 18, 297 abst). Therefore, in order to further investigate the ICSI-mediated method, the present study was conducted to address the integration patterns of foreign genes introduced by this method. In particular, we wished to determine the number of transgene copies and number of chromosomal integration sites. TG pig fetuses, obtained by the ICSI-mediated method in a separate cardiac disease model study, were used in the present study. Porcine cumulus-oocyte complexes that had been collected from slaughterhouse ovaries were subjected to in vitro maturation in NCSU23 medium to produce MII oocytes to be used in this study. Porcine spermatozoa frozen in Beltsville Thawing Solution (BTS) were thawed rapidly in a 37�C water bath, and each spermatozoon was decapitated using ultrasound (28 kHz, 100 W; W-113; Honda Electronics Co., Ltd, Aichi, Japan). The heads (2 to 5 � 105/10 �L) were co-incubated with 2.5 ng �L-1 of rabbit calreticulin cDNA (�MHC-CRT-HA: 7.5 kb) for five min at room temperature, and then microinjected into MII oocytes using a piezo-micromanipulator. An electric stimulus (DC 150 V mm-1, 100 �s) was applied 10 to 40 min after microinjection in order to activate the oocytes. The embryos were cultured in PZM-5 medium for one to two days, and then transferred into the oviducts of recipient gilts, whose estrous cycle had been synchronized using 1000 IU eCG and 1500 IU hCG. Fetuses were collected 33 or 50 days later, and a primary cell line (fibroblast) was established. For each fetus, the number of transgene copies was determined by Southern blot. In addition, the chromosomal sites, where the foreign gene had integrated, were identified, and the number of integration sites was determined by fluoresent in situ hybridization (FISH). A total of 454 ICSI embryos were transferred to 4 recipients (92 to 135 embryos/recipient). All recipients became pregnant and 23 fetuses (5.1%, 23/454), including 7 TG fetuses (30.4%, 7/23), were obtained. Southern blot analysis showed that the number of transgene copies varied between 1 and 300 (1 copy: 1 fetus; 10 copies: 2 fetuses; 30 copies: 3 fetuses; 300 copies: 1 fetus). FISH analysis showed that in TG fetuses, the foreign gene had integrated at only a single chromosomal site, and this site varied from TG fetus to TG fetus. These results demonstrate that, in the case of ICSI-mediated gene transfer, as is the case for gene transfer by pronuclear microinjection, the integration patterns are: multiple copy, random site, and single site integration. This study was supported by PROBRAIN.

ICSI-mediated gene transfer has been used as an alternative method to pronuclear microinjection for the genomic modification of many species. With this method, transgenic embryos are produced by the microinjection of metaphase II oocytes with spermatozoa previously incubated with foreign DNA. Recently, it was shown in mice that the low percentage of transgenic animals produced from injected oocytes, results from the fact that the expression of foreign DNA is associated with paternal chromosome degradation (Szczygiel M.A. et al., 2003 Biol. Reprod. 68, 1903–1910). It is also known that sex chromosomes localize preferentially, at least in humans, on the periphery of the sperm nucleus on sub-acrosomal regions (Sbracia M. et al., 2002 Hum. Reprod. 17, 320–324), suggesting a high level of interaction with foreign DNA molecules with possible impact on the sex ratio of the offspring. In order to test this hypothesis we have compared ICSI (no DNA), and with ICSI-mediated EGFP (5Kb plasmid DNA from Clonetech, Spain) transfer, with ICSI-mediated YRT3 (a mouse tyrosinase gene derivative YAC-DNA with 100Kb; Montoliu L. et al., 1996 EMBO) transfer. Gametes were from 6–8 weeks old CD1 mice. ICSI-mediated gene transfer with post-thawed immotile spermatozoa, extended in M2 medium in the absence of ion chelators (EDTA and EGTA), was done as previously described (Szczygiel M.A. et al., 2003 Biol. Reprod. 68, 1903–1910). Table 1 below summarizes the data collected. Relative to our control, sex ratio deviation was a consequence of the coinjection of DNA. Forty-three percent of males were obtained with regular ICSI, whereas 64% and 65% were the respective percentages when EGFP or YRT3 DNA was coinjected with spermatozoa. This statistically significant (P&amp;lt;0.05, z-test, Sigma Stat, Jandel Scientific, USA) sex ratio deviation, favoring male ICSI offspring when foreign DNA is coinjected, may result from a higher female embryo susceptibility to parental sex chromosome fragmentation induced by the interaction with foreign DNA molecules. Possible impairment of X chromosome inactivation and dosage compensation resulting from the fragmentation of the sex chromosome on X-carrying spermatozoa could explain this female embryo degeneration. Supporting this view, it was recently shown in mice that sex ratio can be skewed against female births by a mutation in a single gene of the X chromosome (Tsix) involved in such mechanisms (Lee J.T., 2002 Nat. Genet.). In conclusion, mouse ICSI-mediated gene transfer induces sex ratio deviation favoring male offspring. Table 1 Sex ratio of the offspring obtained with ICSI, ICSI-mediated EGFP transfer, and ICSI-mediated YRT3 transfer