The techniques for the production of transgenic pigs are described. Once a gene interesting for animal breeding is found, a gene construct is cloned. The gene construct is a combination of a structure gene and a promoter region which regulates the gene expression of the transgene. The DNA solution is microinjected into the pronuclei of fertilized ova. The injected ova are transferred to synchronised recipient pigs. The animals born must be controlled for the integration of the injected gene construct. In transgenic offspring the expression of the gene, the heredity to the next generation and the biological activity have to be tested. The overall efficiency of a gene transfer programme in pig is about 0.5%. This is lower than in similar projects in mice.

The production of the recommended embryonic stage for microinjection of foreign DNA into pronuclei needs a precisely timed preparation of donor pigs. For oestrus induction 1250 IU of PMSG is used at a body weight of 60 kg to 90 kg. To induce ovulation 750 IU of HCG are injected 3 days later. After two inseminations eggs are collected by surgical flush of the oviducts and centrifuged for 3 min at 15,000 g for visualization of the pronuclei. After DNA-microinjection the embryos are transferred to 12 h asynchronous recipients. About 50% of the collected eggs could be used for our experiments. The number and quality of the recovered eggs was related to the body weight of the donors. An in vivo culture system in the non-ligated pig oviduct was established in order to measure the effects of the distinct steps in microinjection on embryo survival. The survival rate of untreated zygotes was 52%. After centrifugation the rate was reduced to 38% and after microinjection to 12%. Pregnancy rate after 98 transfers was 37% and an average of 3.9 piglets were born per litter.

Inhalt: Zur Untersuchung der Auswirkungen auf das Scheidenmilieu wurde 12 Kühen eine PRID‐Spirale in die Scheide eingelegt und dort für die Dauer von 12 Tagen belas‐sen. Vier Tiere wurden mit einliegender Spirale und jeweils 2 am 1. bis 4. Tag nach Entzug der Spirale geschlachtet. Im Untersuchungszeitraum wurden Tupferproben mikrobiologisch und bei der Schlachtung gewonnene Scheidenproben histomorphologisch und histochemisch untersucht. Die Ovulation fand etwa 3 Tage nach dem Ende der Behandlung statt. Zwei weitere Tiere wurden in der Gelbkörperphase mit einem Prostaglandin‐Analog behandelt und 3 Tage darauf geschlachtet. Sie dienten als zyklussynchrone Kontrollen. An den von Behandlungsbeginn bis zur Schlachtung genommenen Tupferproben zeigte sich, daβ die bakterielle Besiedlung von Vagina und Cervix uteri bei den Versuchsgruppen im Untersuchungszeitraum deutlich zunahm. Auch mehrere Tage nach Entzug der Spirale war diese Besiedlung nicht eliminiert. Histologisch lassen sich bei einliegender Spirale unterschiedlich stark akut und chronisch entzündliche Komponenten nachweisen. Epitheldefekte und freie Blutungen sind auf kleine Bezirke beschränkt. Das Epithel ist atypisch und in der Schleimsekretion gestört. Die nach Entzug der Spirale rasch einsetzenden proliferativen Reparationsvorgänge sind am Ende des Untersuchungszeitraumes nicht abgeschlossen. Das Epithel ist, namentlich um den üblichen Besamungszeitpunkt herum, weiterhin nicht zu normaler Schleimsekretion in der Lage, wie sie für den Östrus typkch wäre. Die Untersuchung zeigt, daβ der Einsatz der PRID‐Spirale nicht völlig ohne Probleme ist. Die bakterielle Besiedlung und Störungen in der epithelialen Schleimsekretion sind als Beeinträchtigung des Scheidenmilieus zu werten. 06 hierdurch der Besamungserfolg negativ beeinfluβt wird, bedarf weiterer Abklärung.Contents: Effect of a progesterone releasing intravaginal device (“PRID”) on the intravaginal environment in the cow An experiment was carried out to investigate the effect of the application of “PRID” on histological and bacteriological conditions in the vagina of cows. PRID‐coils (CEVA) were introduced into the vagina of 12 cows where they remained for 12 days. Four cows were slaughtered with the coil in position and 2 cows each were slaughtered on day 1, 2, 3 and 4 after coil removal. Cervical and vaginal mucus collected before introduction and after removal of the coil and at the time of slaughter was used for bacteriological examination. Immediately after daughter the genital tract was removed and tissue samples were taken from different segments of the vagina. Two additional cows were treated with a prostaglandin analogue during the luteal phase. These were slaughtered 3 days later and served as controls. The bacteriological examination revealed that in most cows application of the “PRID”‐coil implies bacteriological contamination of cervix and vagina. Damage to the vaginal epithelium and its secretory activity was histologically evident. Recovery commenced immediately after coil withdrawal but was not complete by the time mating or insemination of cows would normally take place. Further investigation must show if application of the “PRID”‐coil exerts a detrimental effect on the cow's fertility.

The objective of this study was to assess the effect of various aspects of pronuclear DNA microinjection on the early development of porcine ova in utero. Estrus was synchronized and superovulation was achieved in sexually mature gilts by the administration of allyl trenbolone, PMSG and hCG. Donor gilts were bred at 12 and 24 h after the onset of estrus. Ova were recovered between 60 and 62 h after the administration of hCG. One-cell ova that exhibited pronuclei after centrifugation were randomly allocated in equal numbers from each donor across one of two pairs of treatments: micro-DNA (ova were injected with two gene constructs that code for the human complement regulatory proteins decay accelerating factor and membrane cofactor protein) and control (ova were centrifuged only) or micro-buffer (ova were injected with buffer only) and pierced (a pipette was inserted into one pronucleus). Ova were transferred by treatment pairs to recipients. Treatments were segregated by oviduct. Ova were recovered after 120 h in utero, fixed and stained with 1% orcein. The proportion of ova that possessed ≥80 nuclei, the mean number of nuclei present and proportion of ova that formed blastocysts were all significantly (P<0.05) greater for control and pierced ova than for micro-DNA and micro-buffer ova. No difference in these parameters was observed between micro-DNA and micro-buffer ova. These results demonstrate that pronuclear microinjection of a buffer alone can adversely affect the early development of porcine ova in utero.

We compared in vitro survival rate of mouse embryos after cryopreservation in phosphate buffered saline (PBS) with that in Hepes buffered Tyrode's (HeTy) medium. We also examined the effect of size of straw (0.25 ml versus 0.50 ml) in which embryos were frozen on the in vitro survival post thawing. Early to mid-morula (n=823) from superovulated females were randomly assigned to 1 of 4 treatment groups: Group 1 = PBS in 0.25-ml straws, Group 2 = PBS in 0.50-ml straws, Group 3 = HeTy in 0.25-ml straws, Group 4 = HeTy in 0.50-ml straws. Embryos exposed to glycerol in 3 steps (0.44 M, 0.88 M and 1.4 M) at 22°C were loaded into straws and placed in a programmable freezer at −6°C, manually seeded during a 6 min holding period and cooled to −34°C at 0.5°C/min. After a 15-min holding period at −34°C, the embryos were plunged into liquid nitrogen. Thawing was done by exposing each straw to air (22°C) for 10 sec before plunging into a 37°C water bath for 15 to 20 sec. Embryos were rehydrated for 10 min at 37°C in 0.8 M sucrose, washed (3 times) and cultured for 72 h in modified Ham's F-10 + 10% fetal calf serum (FCS) at 38°C in 5% CO2/air. The percentages of expanding blastocysts at 72 h were 68% (140206), 68% (135197), 77% (164212) and 69% (143208) for Groups 1, 2, 3 and 4, respectively. The survival rate of embryos frozen in HeTy (Groups 3 and 4) was slightly higher (73%; P = 0.07) than those frozen in PBS (68%, Groups 1 and 2). Straw size did not have an affect (P = 0.19) on post-thaw development, nor was there an interaction between media and straw size (P = 0.13). We conclude that mouse embryos can be successfully frozen in either 0.25- or 0.50-ml straws, and that HeTy is a suitable medium for mouse embryo cryopreservation; however, further studies with embryos of other species are warranted.

Reproduction in Domestic AnimalsVolume 20, Issue 4 p. 251-252 Production of transgenic mice, rabbits and pigs by microinjection into pronuclei R.C. Selden, R.C. Selden LMU, München, FRG, Massachusetts General Hospital, Boston, USASearch for more papers by this authorK. Springman, K. Springman Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorJ. Hondele, J. Hondele Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorJ. Meyer, J. Meyer Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorE.-L. Winnacker, E.-L. Winnacker Institut für Biochemie, Boston, USASearch for more papers by this authorH. Kräußlich, H. Kräußlich Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorG. Brem, G. Brem Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorB. Brenig, B. Brenig Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorH. M. Goodman, H. M. Goodman LMU, München, FRG, Massachusetts General Hospital, Boston, USASearch for more papers by this authorF. Graf, F. Graf Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorB. Kruff, B. Kruff Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this author R.C. Selden, R.C. Selden LMU, München, FRG, Massachusetts General Hospital, Boston, USASearch for more papers by this authorK. Springman, K. Springman Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorJ. Hondele, J. Hondele Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorJ. Meyer, J. Meyer Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorE.-L. Winnacker, E.-L. Winnacker Institut für Biochemie, Boston, USASearch for more papers by this authorH. Kräußlich, H. Kräußlich Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorG. Brem, G. Brem Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorB. Brenig, B. Brenig Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorH. M. Goodman, H. M. Goodman LMU, München, FRG, Massachusetts General Hospital, Boston, USASearch for more papers by this authorF. Graf, F. Graf Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this authorB. Kruff, B. Kruff Institut für Tierzucht und Tierhygiene, Boston, USASearch for more papers by this author First published: December 1985 https://doi.org/10.1111/j.1439-0531.1985.tb00423.xCitations: 124AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Hammer, R.E., V.G. Pursel, C.E. Rexroad Jr., R.J. Wall, D.J. Bolt, K.M. Ebert, R.D. Palmiter, R.L. Brinster, 1985, Nature 315, 680–683. 10.1038/315680a0 CASPubMedWeb of Science®Google Scholar 2 Palmiter, R.D., R.L. Brinster, R.E. Hammer, M.E. Trumbauer, M.G. Rosenfeld, N.C. Birnberg, R.M. Evans, 1982, Nature 300, 611–615. 10.1038/300611a0 CASPubMedWeb of Science®Google Scholar 3 Palmiter, R.D., G. Norstedt, R.E. Gelinas, R.E. Hammer, R.L. Brinster, 1983, Science 222, 809–814. 10.1126/science.6356363 CASPubMedWeb of Science®Google Scholar Citing Literature Volume20, Issue4December 1985Pages 251-252 ReferencesRelatedInformation