Fundamental Study On Creating A Transgenic Dog

Canine is one of the most important laboratory animals in the biomedicine research. Canine entire genome atlas was completed at the end of 2005 year. Compared with other animals, such as mouse, canine gene was much more similar to human gene. About 360 kinds of canine genetic diseases are similar to human's. Therefore, the gene knock-in or gene knock-out dog was one of the ideal animal models to study human gene functions and the genetic diseases. People had succeeded in creating more than 10 of kinds transgenic animals such as mouse, rat, cavy, hamster, cattle, sheep, pig, rabbit and so on. More and more transgenic animals had been applied to the biological science research. Progress is slow on canine reproductive biology because of the specificity of its reproductive organs and reproductive physiology. The transgenic dog still hasn't been reported by now. This dissertation demonstrated the basic research on creating the transgenic dog. This paper is divided into three parts.1. Study on the reproductive biology of bitchesWe investigated 463 Beagle bitches/parturitions for 3 years, which were bred in Yangzhou and Suzhou respectively. We discovered that the bitches could be in estrous at all the year round, but especially in spring and autumn. The bitches were monoestrous. The average interval time of oestrus was 34 weeks. But the percent of the estrous bitches had certain differences in each season. The oestrus rate of the spring and autumn was 71% and 72.8% respectively on the two farms, which indicated that the bitch wasn't the classic seasonal oestrus animal. The gestation period was 62.9 days on average, mostly between 61～64 days. Each bitche gave birth to 5.35 puppies on average each time, mostly between 3～7 puppies and sometimes up to 10 puppies. There was no significant difference in the reproductive performance between the two farms.By dissecting the bitches' generative organs, we could see that the ovary was packaged by the bursa ovarica. The bent and short oviduct was in the bursa ovarica's parietes. Along with the follicle's growth and development, the ovary's surface showed a petal structure. The histological research on the ovary of postnatal bitches demonstrated as following: Ovarian cortex sections of newborn bitches had large numbers of primordial follicles. With the age increasing, the primary follicles appeared in 3 mounth-old, the secondary follicles appeared in 6 month-old and the mature follicle appeared in 9 month-old. The endometrial gland developed and proliferated continuously after newborn, then gradually developed. At the same time, the stroma became denser. The oviduct had formed primary mucosal folds from newborn to 9 month-old, lots of secondary mucosal folds emerged after 12 months.The dynamic change of estradiol (E₂) and progesterone (P) in bitches' serum was tested by radioimmunoassay (RIA) to bitches in puberty and estrous cycle. The results as following: The E₂ concentration was extremely low at birth, then ascended gradually in 3 month-old and maintained constantly about 20pg/ml after 9 month-old. The P concentration was zero in the newborn, but later increased and stayed at a lower level ( < 0.6 ng/ml). The concentration of E₂ and P both maintained at a lower level during the anestrus. At the onset of proestrus, E₂ increased gradually, then reached the first peak (75pg/ml) in the 4th day before mating, but later decreased to some extent. Until the 18th days after pregnancy, the second peak appeared due to the secretion of corepus luteum. P rised slowly in the early stage of proestrus, but rapidly in the later stage. It increased from 2.7ng/ml of the 4th day before the mating to 10.9ng/ml in the mating, later increased continually. The 14th day after the pregnancy, it got to the peak 39.4 ng/ml and later maintained for a period of time. P declined gradually from the end stage of pregnancy to the parturition, and it reach 1ng/ml below in the parturition.The method of in situ hybridization (ISH) was first applied to detect relative expression abundance of estrogen receptor a (ERα) mRNA and progesterone receptor (PR) mRNA both the reproductive organ and the pituitary gland of the bitches. This study explained their relativities between sex hormonal concentration in serum, morphopoiesis of reproductive organ and expression abundance of ERa and PR mRNA. It laid the foundation to further explore the relationship between the reproductive capacity and the difference in the level of ER and PR gene transcription and translation. Results of image manipulation analysis system indicated that ER mRNA almost are not detected in the ovary at birth. ER mRNA expression increased from 3rd to 9th month-old, and then declined slightly. Postive staining was mainly detected in the granule cells of the various follicles, thicker in big follicles than in small follicles. Ovary expression of ERamRNA remained high during the proestrous and estrous . In uterus, ERamRNA was mainly detected in endometrium gland and lesser in the stroma. In estrus cycle, ERamRNA expression showed a downward trend. ERamRNA was detected mainly in the pituitary gland cells. ERamRNA were almost not detected at birth. With the increase of age, ERamRNA had been maintained at a higher level. In the estrous cycle, ERamRNA expression showed a downward trend in the pituitary.Above results of canine reproductive biology is help for improving canine fertility and arranging the experimentates reasonably. What's more, they have provided the theory evidence of inducing estrus, superovulation of dogs and so on.2. Related study on the assisted reproductive technology for creating a transgenic dog with the femaleThe research was based on the feminity to creating a transgenic dog. First, lots of zygotes were needed. Actually the artificial inducing estrus of bitch was extremely difficult now, due to ovaries were packaged by bursa ovarica ,and oocytes' recovery was unable by laparoscope. In vitro maturation and artificial inducing estrus were crucial assisted reproductive technologies for canine, which was the important way to create transgenic animals. But at present, the canine ART cann't satisfy the need of the transgenic dog production. This study was concern with in vitro oocytes maturation, inducing estrus and embryo transfer in oviduct of bitch. The embryo transfer in oviduct technology established a platform for generating transgenic animal.Massive procaryon zygotes were required in embryo operatation. We sliced ovaries to release immature oocytes. We had established the oocyte culture system. We examined the number of good oocytes collected from bitches ovaries at diffenent ages and various stages of the estrous cycle by slicing the ovaries, then cultured them to do comparative experiment in vitro with the different culture medium additive, such as serum, EGF and E₂, which could promote the oocytes maturation. The standard of oocytes maturation was achieving MII by using hoechest 33342 staining technique. Results demonstrated that, the recover-ratio of COCs in proestrus and oestrus was higher than other periods. The average returning quantity from 3-year-old bitches was the highest, but <1 and >7-year-old bitches was the lowest. The nuclear maturation rates of oocytes in the culture medium supplmented with EGF and E₂ were higher than that supplemented with EGF or E₂ and the control (22.58% vs 18.00%, 5.56%; 22.58% vs 4.76%). There's no difference in the maturation rates of oocytes between 20% FBS and 20% EDS trial (17.14% vs 17.95%), which were higher than the control (0%). EGF had effect on cumulus expansion, but the rate of oocytes was not depend on the EGF dose. It showed that 72h was suitable for IVM of canine oocytes. The cumulus cells had important effect on oocytes maturation, as the oocytes with cumulus had higher maturation rates than oocytes without cumulus (15.56% vs 3.13%).We carried out experiments of inducing estrus of anoestrum, young of nonestrus and sterile agedness bitches by i.m. PMSG + hCG. The results as following: extraneous hormone (1200IU PMSG + 500IU hCG) could induce effectively the anoestrus and young of nonestrus bitches to estrous, but useless for the sterile agedness dogs. In an invariable dose PMSG 1200IU, the inducing estrus rate was 40% (6/15) by given medicine once, but that was 50% (4/8) in the anoestrus group. That was 50% (15/30) by given medicine several times but a little ounce each time, the inducing estrous rate was 69% (11/16). Furthermore, The deliver puppies rate of bitches was 31% (5/16) . These bitches' hormone variation rule was the same as generally breeding bitches'. The results showed that this way was feasible to supply with more estrous bitches.Embryo transfer was also one of the assisted reproductive technologies. We transfer zygotes of 1-8 cells stage into 12 recipient's oviducts. There were 3 receivers giving birth to 6 puppies at the 61th-65th day after the donor's oocytes fertilization, the successful rate was 25%(3/12). it was the first to obtain living puppies in this way. 3. Some fundamental research to create a transgenic dog based on maleCreating the transgenic animals is mediated by the Spermatogonial stem cell (SSCs), including transgenosis in vivo and in vitro. The latter included SSCs culture in vitro, eliminating endogenous SSCs, SSCs transfer and so on. Testes were collected by surgery operation from 4-5 month-old Beagle dogs. The SSCs were isolated by enzymatic combinations. According to the differentiation of velocity of cells sticking to the wall in vitro, SSCs were purified and then cultivated with DMEM containing different concentration of serum or EGF respectively. By counting the clusters every day, the growth efficiency of SSCs can be readily detected within 1 week in a semi-quantitative manner. The SSCs were transfected with pEGFP-N1 plasmids by the cationic polymer transfection reagen and calcium phosphate transfection reagen. The results as following: collagenase IV+ trypin combination was an effective method to dissociate canine SSCs, the total number of dissociated cells was 6.67×10⁶/100mg testis parenchyma and rate of living SSCs was 96.5%. It Is necessary to add FBS to the culture medium for SSCs culture in vitro . Compared with EGF group, the clusters of cell in the 20ng/ml group increased markedly. The gene transfection efficiency of the cationic polymer transfection reagen method was much higher than that of calcium phosphate method (22.1% vs 8.1%) .In conclusion, we had developed an efficient and economical method to isolate the SSCs, through which we could obtain enough viable germ cells. The technique of SSCs culture demonstrated that dog SSCs could be maintained in culture medium in vitro for approximately 1 week. The clusteres came from a single cell indicated that SSCs are able to achieve growth and proliferation in the culture system in vitro. We have succeeded in the EGFP gene transfection in vitro, which was prepared for the further SSCs transplantation technique to get the transgenic puppies.