In order to optimize the overall efficiency of pronuclear microinjection, we designed experiments to: 1) test the best developmental stage for transferring injected embryos to obtain pregnancies and transgenic pups; 2) determine the optimum number of injected embryos transferred to obtain pregnancies and transgenic pups; 3) investigate whether addition of non-injected embryos with injected embryos increased pregnancy rate (PR) and transgenic pups; and 4) establish the time during pregnancy of highest embryonic or fetal loss. Mice (CD1; 3 to 4 wk old), were superovulated with 10 iu PMSG and 5 iu hCG 48 h apart. One-cell embryos were collected for microinjection 20 to 24 h after hCG. The gene used was the whey acidic protein promoter linked to a coding sequence of the human protein C gene (WAP-hPC). Embryos were cultured in CZB at 37°C in 5% CO₂ in air. All the live pups born and embryos and fetuses recovered were analyzed by the polymerase chain reaction to detect the presence of the transgene. Experiment one consisted of nine transfer treatments (TRT) which included all the combinations of three developmental stages (1-cell, 2-4 cell and morula/blastocyst) with three quantities of embryos per transfer (15-24, 25-34 and 35-44). Ten transfers were performed for each TRT. The highest PR and total pups born (TOTP) were obtained after transferring 25 to 34 2-4 cell embryos (PR=90% TOTP=3.5/ pregnancy). However, overall analysis indicated that the percentage of transgenic pups born (%TRS) was highest using 1-cell embryos [33.9%, 20.0% and 11.1% for 1-cell, 2-4 cell and morula/blastocyst (mor/bl), respectively]. The second experiment consisted of six transfer TRT: 20-0, 16-4, 12-8,30-0, 26-4, and 22-8 injected - non-injected embryos, respectively (10 transfers/TRT). Data showed that PR and TOTP can be improved by addition of non-injected embryos. However, the percentage of transgenic pups was significantly (p< .05) higher when all the embryos transferred were injected (53.6 % vs 46.4 % for transfers without and with non-manipulated embryos, respectively). Additionally, 30 embryos per transfer yielded a significantly higher percentage (p< .05) of transgenic pups than 20 embryos per transfer (67.9 % vs 32.1 % for 30 and 20 embryos per transfer, respectively). In experiment three 45 transfers of microinjected embryos were performed (30 embryos per transfer). Fifteen recipients were sacrificed on day 4, 12, and 18 of gestation. On each day all embryos and fetuses were counted and analyzed for the presence of the transgene. The percentage of transgenic embryos or fetuses was not statistically different at any recovery day (45.8%, 35.5%, and 34.6% for days 4, 12, and 18, respectively). However, the number of viable embryos at day 4 was significantly greater than the number of viable fetuses on days 12 or 18 (10 ± 1.1,,5.1 ± 1.6, and 2.4 ± 1.3 for days 4,,12 and 18, respectively). Collectively, the results indicate that: 1) transfer of 20 to 30 1-cell embryos was the best method to obtain transgenic mice, 2) addition of non-injected embryos decreased the number of transgenic pups obtained per pregnancy, and 3) although most of the embryonic losses after microinjection happen before day 4 of gestation, additional losses occurred between days 4 and 18 of pregnancy. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/37502 |
| Date | 17 March 2010 |
| Creators | Canseco-Sedano, Rodolfo |
| Contributors | Animal Sciences, Dairy, Gwazdauskas, Francis C., Pearson, Ronald E., Eng, Ludeman A., Nebel, Raymond L., Velander, William H., Vinson, William E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | ix, 80 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 29179369, LD5655.V856_1992.C367.pdf |
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