Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.     

Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology

Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 ± 4.1 and 24.5 ± 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.     

Survival of early preimplantation porcine embryos after co-culture with cells producing an avian retrovirus

To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.     

Aberrant fetal growth and development after in vitro culture of sheep zygotes.

The effects of in vitro culture systems for sheep zygotes on subsequent fetal growth and development to day 61 and day 125 of gestation were studied. Zygotes recovered from superovulated Scottish Blackface ewes approximately 36 h after intrauterine insemination using semen from a single Suffolk sire were cultured for 5 days in (a) a granulosa cell co-culture system (co-culture); (b) synthetic oviductal fluid medium without serum (SOF-); and (c) synthetic oviductal fluid medium supplemented with human serum (SOF+). Control embryos were recovered from superovulated donor ewes at day 6 after oestrus. Embryos were transferred at day 6 to synchronous Scottish Blackface recipient ewes. In total, 146 gravid uteri were recovered, comprising 97 at day 61 (20 co-culture, 27 SOF-, 25 SOF+ and 25 control) and 49 at day 125 (13 co-culture, 8 SOF-, 6 SOF+ and 22 control) of gestation. Fetuses derived from co-cultured embryos were 14% heavier (P < 0.01) by day 61 of gestation than those derived from control embryos. Growth coefficients derived from the linear allometric equation logey = logea + b logex (where y = organ mass; x = fetal mass) were significantly greater (P < 0.05) for liver, heart, kidneys and plantaris muscle in fetuses derived from co-cultured embryos, and for liver in fetuses derived from SOF+ embryos than those for control fetuses. Fetuses derived from co-cultured embryos were 34% heavier (P < 0.001) and fetuses derived from SOF+ embryos were 18% heavier (P < 0.01) by day 125 of gestation than those derived from control embryos. Growth coefficients for liver and heart for fetuses derived from co-culture and SOF+ embryos were also significantly greater (P < 0.05) at this stage of gestation than those for control group fetuses. In contrast, allometric coefficients for these organs in fetuses derived from embryos cultured in SOF without serum supplementation were not different from those for controls. Excessive volumes of amniotic fluid (polyhydramnios) were observed in 23% of conceptuses derived from co-cultured embryos. In vitro embryo culture can significantly influence fetal growth and this study provides quantitative evidence of major shifts in the patterns of organ and tissue development.     

Evaluation of gestational deficiencies in cloned sheep fetuses and placentae.

Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.     

Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin

A total of 511 embryos was recovered non-surgically from nearly 100 superovulated or untreated donors. Superovulation with FSH-LH resulted in more corpora lutea, recovered ova, and pregnancies (P<.01) than superovulation with PMSG. No differences were observed in numbers of ovulations, embryos recovered, or pregnancies per donor when prostaglandin F_2α was given to donors 2 versus 3 days following gonadotrophin treatment.Pregnancy rates of 12, 31, 58, and 63% were obtained from groups of embryos classified morphologically as poor, fair, good, and excellent (P<.05). Morphologically normal embryos collected and transferred at 5 to 6 days of gestation resulted in more (P<.05) fetuses (75%) than morphologically normal embryos at 8 to 9 days of gestation (56%), but neither was significantly different from morphologically normal embryos at 6.5 to 7.5 days of gestation (61%). There was no difference (P>.05) between pregnancy rates when retarded embryos from untreated donors (12%) were compared to retarded embryos from superovulated donors (22%). However, a higher proportion of morphologically normal embryos from untreated donors developed into fetuses (71%) than did morphologically normal embryos from superovulated donors (59%, P<.05).     

Brain abnormalities induced by murine cytomegalovirus injected into the cerebral ventricles of mouse embryos exo utero

Murine cytomegalovirus (MCMV) was injected into the cerebral ventricle of mouse embryos on day 13 of gestation after exposing the embryos out of the uterus in the abdominal cavity of the mother. The embryos were allowed to develop to day 18 of gestation, then taken out from the abdominal cavity. Macroscopically, there were four expanded and three distorted brains out of 19 surviving embryos, whereas no brain abnormality was noticed in 13 embryos injected with culture medium instead of MCMV in the same way. Histopathological examination showed hydrocephalic lesions with strong dilatation of the ventricles and atrophy of the cerebral cortex, and inflammatory lesions with granulomatous proliferation of the ventricular walls with disappearance of the cortical zonation. Immunohistochemically, MCMV-induced nuclear antigen-positive cells were frequently observed in the wall of the ventricles and occasionally scattered in the cerebral cortex, white matter, and the nucleus basalis. Some fetuses injected with MCMV in the same way were recovered from the abdominal cavities on day 18 of gestation and transferred to foster nurse mothers. They showed massive cerebral necrosis after feeding for 9 days after birth. Brain abnormalities of mouse embryos after intraventricular injection with MCMV may provide an experimental model of brain damage induced by congenital cytomegalovirus infection.     

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.     

Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster.

The extremely high rate of cell division that occurs during early embryogenesis is hypothesized to predispose to high rates of mutation after chemical exposure. We tested this supposition experimentally. To probe the variation in susceptibility to mutation induction as a function of gestation stage, somatic cells of the developing Syrian hamster were isolated after transplacental treatment with N-ethyl-N-nitrosourea (ENU). Mutants were quantified using either 6-thioguanine (6-TG) or diphtheria toxin (DT) as selective agents. Several different approaches were used. In one, three litters were exposed on each gestation day and fetuses were removed on day 13. Maximum fetal sensitivity to ENU's genotoxic action was noted when treatment was at days 8 and 9, fewer mutants being obtained with earlier and later exposures. To compensate for the low numbers of target cells early in gestation, this experiment was repeated using larger numbers of litters exposed at the earlier time points, and the highest mutation frequency was now found to occur after treatment on gestation days 6 and 7. In the second approach, mutations were quantified in cells harvested 24 h after transplacental ENU exposure. Here again, embryos exposed at earlier times of gestation were more susceptible than those treated at later periods. Based on the total cell numbers in embryos and fetuses at each gestation day, we conclude that mutation frequency is maximal on day 6, corresponding to the primitive streak stage with extremely high rates of cell division.     

Transgenic sheep generated by lentiviral vectors: safety and integration analysis of surrogates and their offspring

The safety of HIV-1 based vectors was evaluated during the production of transgenic sheep. Vectors were introduced into the perivitelline space of in vivo derived one-cell sheep embryos by microinjection then transferred into the oviducts of recipient females. At 60–70 days of gestation, a portion of the recipients were euthanized and tissues collected from both surrogates and fetuses. Other ewes were allowed to carry lambs to term. Inadvertent transfer of vector from offspring to surrogates was evaluated in 330 blood and tissue samples collected from 57 ewes that served as embryo recipients. Excluding uterine contents, none of the samples tested positive for vector, indicating that that the vector did not cross the fetal maternal interface and infect surrogate ewes. Evaluating ewes, fetuses and lambs for replication competent lentivirus (RCL); 84 serum samples analyzed for HIV-1 capsid by ELISA and over 600 blood and tissue samples analyzed by quantitative PCR for the VSV-G envelopes revealed no evidence of RCL. Results of these experiments provide further evidence as to the safety of HIV-1 based vectors in animal and human applications.     

In vivo development of vitrified rabbit embryos: Effects on prenatal survival and placental development

The aim of this work is to study the effect of the vitrification procedure on prenatal survival and on placental development at the end of gestation in rabbits (Oryctolagus cuniculus). One hundred eighty-one females were slaughtered at 72 h of gestation. Morphologically normal embryos recovered at 72 h of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos (320 embryos) were transferred into a total of 24 does and fresh embryos (712 embryos) were transferred into a total of 43 does. Females were induced to ovulate 72 h before transfer when fresh embryos were transferred and 60 to 63 h before transfer when vitrified embryos were transferred. Each recipient doe received eight embryos into the left oviduct and eight embryos into the right oviduct. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at Day 14 of gestation. Recipient females were slaughtered by stunning and exsanguination 25 d after the transfer, and fetuses were classified according to their status. Live fetuses and fetal and maternal placenta were weighed Pregnancy rate was defined as the total number of females having at least one live fetus at Day 28 of gestation divided by the total number of females. Prenatal survival was estimated as live fetuses at Day 28 of gestation divided by the number of transferred embryos. The pregnancy rate after transfer of vitrified embryos (92%) was similar to that achieved with fresh embryos (86%), but prenatal survival was lower for vitrified than for fresh embryos (53% vs. 34%). We did not find differences in embryo survival from 72 h to implantation. Transfer of vitrified embryos reduced fetal survival from implantation to Day 28 (57% vs. 82%). Differences in the number of live fetuses at Day 28 of gestation were mainly due to the higher fetal mortality observed soon after implantation in pregnancies resulting from the transfer of vitrified embryos. A higher percentage of decidual reactions and atrophic maternal placentas (27.5% vs. 8.3%) and also of atrophic fetal and maternal placentas (12.1% vs. 5.3%) were observed after transfer of vitrified embryos. Both treatments showed similar percentage of dead fetuses (3.3% vs. 4%). Maternal placenta of the fetuses from fresh embryos was 15% heavier than maternal placenta of fetuses from vitrified embryos.     

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.     

Influence of in vitro systems on embryo survival and fetal development in cattle

In vitro systems are commonly used for the production of bovine embryos. Comparisons between in vivo and in vitro produced embryos illustrate that the morphology of preimplantation-stage embryos differ significantly, the survival of embryos and fetuses is decreased, the size distributions of the populations of conceptuses and fetuses are altered throughout gestation, and placental development is significantly changed. Taken together these findings indicate that exposure to some in vitro environments during the first 7 days of life can profoundly influence fetal and placental development in cattle. An understanding of how in vitro oocyte maturation, in vitro fertilization, and embryo culture systems influence both fetal and placental development should result in systems that consistently produce normal embryos, fetuses, and calves.     

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18–22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (α-antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8 to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines. © 1996 Wiley-Liss, Inc.     

Viable Transgenic Goats Derived from Skin Cells

The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.     

Development of reconstituted embryos derived from transgenic embryonic stem cell nuclei

This study was carried out to transform embryonic stem (ES) cells and to produce the reconstituted embryos derived from transgenic ES cell nuclei. Then, in vitro/vivo developmental potency of transgenic ES cells were compared to that of control ES cells (non-transgenic ES cells) in the reconstituted embryos. Unfertilized B6D2F1 ooplasm at metaphase II (M II) and two kinds of ES cell lines, 129SV and transgenic (tg) 129SV transformed by EGFP gene, were used as nuclear recipients and nuclear donors, respectively. The M II chromosome-spindle complex was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, the ES cell nuclei was injected into the enucleated ooplasm directly. Then, reconstituted embryos were activated in SrCl2, and they were cultured in HTF medium. There was no difference of developmental rate between reconstituted embryos derived from the control (non-transgenic) and the tg ES cells. From this result, we indicated that transgenic ES cells might not change the property of peculiarity of the ES cell by gene transfer. The expression of GFP gene in the embryos was observed by fluorescence microscope at the 4-cell and more stage. As comparison with development of the embryos derived from the control and tg ES cells, the difference of the development could not be confirmed between the two cell groups. When the reconstituted embryos derived from the control and tg ES cells were transferred into oviduct or uterus of pseudopregnant females, fetuses were observed 13.5 days post coitum. However, in all fetuses, developmental arrest and regression were seen 19.5 days post coitum.     

Biochemical changes in the developing rat central nervous system due to hyperthermia

Rat embryos at 10 days of gestation were exposed to 43 degrees C for 8 minutes by submerging the exteriorized right uterine horn in heated saline solution and then reinserting the uterine horn into the abdominal cavity. At 15 days, the fetuses were removed, and cells from the cerebral hemispheres were dissociated and grown as primary cultures. Embryos from the left uterine horn served as controls. No morphological changes were observed between the cultures of cells from control and heat-exposed embryos at different days in culture. However, exposure of embryos to hyperthermia at 10 days significantly affected the developmental pattern of activities of acetylcholine esterase associated with cholinergic neurons and of 2',3'-cyclic nucleotide phosphohydrolase associated with oligodendrocytes and myelin membrane formation. These results suggest that hyperthermia at 10 days of gestation in the rat may lead to an impairment in the development of neurons and oligodendrocytes in the central nervous system.     

Generation of transgenic cattle by lentiviral gene transfer into oocytes

The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.     

Prediction of Transgene integration by Noninvasive Bioluminescent Screening of Microinjected Bovine Embryos

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 °C, 15 min) and 4–6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.