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Xue F.,Renova Life, Inc. | Ma Y.,Yale University | Chen Y.E.,University of Michigan | Zhang J.,University of Michigan | And 9 more authors.
Cellular Reprogramming | Year: 2012

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. © Copyright 2012, Mary Ann Liebert, Inc. 2012.


Du F.,Nanjing Normal University | Du F.,Renova Life, Inc. | Chen C.-H.,Renova Life, Inc. | Li Y.,Nanjing Normal University | And 7 more authors.
Cellular Reprogramming | Year: 2015

The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified-thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. © Copyright 2015, Mary Ann Liebert, Inc.


An L.,Nanjing Normal University | Chang S.,Nanjing Normal University | Hu Y.,Nanjing Normal University | Li Y.,Nanjing Normal University | And 6 more authors.
Cryobiology | Year: 2015

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-μL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P > 0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P< 0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P< 0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P > 0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P > 0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P < 0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages. © 2015 Elsevier Inc.


An L.,Nanjing Normal University | Ling P.P.,Nanjing Normal University | Zhu X.,Nanjing Normal University | Liu Y.,Nanjing Normal University | And 11 more authors.
Reproduction in Domestic Animals | Year: 2016

The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo-derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo-derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle-stimulating hormone (FSH) sources, Folltropin® Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well-established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo-derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients. © 2016 Blackwell Verlag GmbH.


An L.,Nanjing Normal University | Chang S.,Nanjing Normal University | Hu Y.,Nanjing Normal University | Li Y.,Nanjing Normal University | And 5 more authors.
Cryobiology | Year: 2015

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3. min followed by immersion in vitrification solution for 30-45. s, and then three embryos per 3-μL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P >. 0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P <. 0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P <. 0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P >. 0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P >. 0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P <. 0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages. © 2015 Elsevier Inc.


PubMed | ARSIAL, Renova Life, Inc., Lannuo Biotechnologies Wuxi Inc. and Nanjing Normal University
Type: Journal Article | Journal: Cryobiology | Year: 2015

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-L vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P>0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P<0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P<0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P>0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P>0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P<0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.


PubMed | ARSIAL, Lannuo Biotechnologies Wuxi Inc. and Nanjing Normal University
Type: | Journal: Scientific reports | Year: 2016

We investigated the effects of 5-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7-2 vs. F7-2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7-1 vs.F7-1, 12.1 vs. 23.6%; Site Three, tF7-3 vs.F7-3, 7.7 vs 10.9%) (P<0.05). Out of 15 predicated off-target sites, tru-RGNs showed significantly decreased frequencies at 5 sites. By microinjecting tru-RGN RNAs into zygotes, FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P<0.05), but not at Site three (39.4 vs 27.8%) (P>0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.


PubMed | ARSIAL, Renova Life, Inc., Instituto Superiore Of Sanita, Lannuo Biotechnologies Wuxi Inc. and Nanjing Normal University
Type: Journal Article | Journal: Reproduction in domestic animals = Zuchthygiene | Year: 2016

The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo-derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo-derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle-stimulating hormone (FSH) sources, Folltropin() Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well-established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo-derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.

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