Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) ho...
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Autor*in:	Meng, Fanli [verfasserIn] Li, Hui [verfasserIn] Wang, Xiaoli [verfasserIn] Qin, Guangsheng [verfasserIn] Oback, Björn [verfasserIn] Shi, Deshun [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2015

Schlagwörter:	Buffalo Cytoplasmic injection Plasmid Transgenic Zygote

Übergeordnetes Werk:	Enthalten in: Journal of animal science and biotechnology - Beijing, 2010, 6(2015), 1 vom: 20. Okt.
Übergeordnetes Werk:	volume:6 ; year:2015 ; number:1 ; day:20 ; month:10

Links:	Volltext

DOI / URN:	10.1186/s40104-015-0044-x

Katalog-ID:	SPR032858035

Internformat


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245	1	0	\|a Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
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337			\|a Computermedien \|b c \|2 rdamedia
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520			\|a Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.
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650		4	\|a Cytoplasmic injection \|7 (dpeaa)DE-He213
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700	1		\|a Li, Hui \|e verfasserin \|4 aut
700	1		\|a Wang, Xiaoli \|e verfasserin \|4 aut
700	1		\|a Qin, Guangsheng \|e verfasserin \|4 aut
700	1		\|a Oback, Björn \|e verfasserin \|4 aut
700	1		\|a Shi, Deshun \|e verfasserin \|4 aut
773	0	8	\|i Enthalten in \|t Journal of animal science and biotechnology \|d Beijing, 2010 \|g 6(2015), 1 vom: 20. Okt. \|w (DE-627)669003441 \|w (DE-600)2630162-3 \|x 2049-1891 \|7 nnns
773	1	8	\|g volume:6 \|g year:2015 \|g number:1 \|g day:20 \|g month:10
856	4	0	\|u https://dx.doi.org/10.1186/s40104-015-0044-x \|z kostenfrei \|3 Volltext
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912			\|a GBV_ILN_62
912			\|a GBV_ILN_63
912			\|a GBV_ILN_65
912			\|a GBV_ILN_69
912			\|a GBV_ILN_70
912			\|a GBV_ILN_73
912			\|a GBV_ILN_74
912			\|a GBV_ILN_90
912			\|a GBV_ILN_95
912			\|a GBV_ILN_100
912			\|a GBV_ILN_105
912			\|a GBV_ILN_110
912			\|a GBV_ILN_120
912			\|a GBV_ILN_121
912			\|a GBV_ILN_138
912			\|a GBV_ILN_150
912			\|a GBV_ILN_151
912			\|a GBV_ILN_152
912			\|a GBV_ILN_161
912			\|a GBV_ILN_170
912			\|a GBV_ILN_206
912			\|a GBV_ILN_213
912			\|a GBV_ILN_224
912			\|a GBV_ILN_230
912			\|a GBV_ILN_285
912			\|a GBV_ILN_293
912			\|a GBV_ILN_370
912			\|a GBV_ILN_374
912			\|a GBV_ILN_602
912			\|a GBV_ILN_647
912			\|a GBV_ILN_702
912			\|a GBV_ILN_2001
912			\|a GBV_ILN_2003
912			\|a GBV_ILN_2005
912			\|a GBV_ILN_2006
912			\|a GBV_ILN_2007
912			\|a GBV_ILN_2008
912			\|a GBV_ILN_2009
912			\|a GBV_ILN_2010
912			\|a GBV_ILN_2011
912			\|a GBV_ILN_2014
912			\|a GBV_ILN_2015
912			\|a GBV_ILN_2018
912			\|a GBV_ILN_2020
912			\|a GBV_ILN_2021
912			\|a GBV_ILN_2025
912			\|a GBV_ILN_2026
912			\|a GBV_ILN_2027
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912			\|a GBV_ILN_2044
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912			\|a GBV_ILN_2055
912			\|a GBV_ILN_2056
912			\|a GBV_ILN_2057
912			\|a GBV_ILN_2059
912			\|a GBV_ILN_2061
912			\|a GBV_ILN_2064
912			\|a GBV_ILN_2065
912			\|a GBV_ILN_2068
912			\|a GBV_ILN_2088
912			\|a GBV_ILN_2093
912			\|a GBV_ILN_2110
912			\|a GBV_ILN_2111
912			\|a GBV_ILN_2113
912			\|a GBV_ILN_2119
912			\|a GBV_ILN_2129
912			\|a GBV_ILN_2147
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912			\|a GBV_ILN_2152
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912			\|a GBV_ILN_2507
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912			\|a GBV_ILN_4277
912			\|a GBV_ILN_4305
912			\|a GBV_ILN_4306
912			\|a GBV_ILN_4307
912			\|a GBV_ILN_4313
912			\|a GBV_ILN_4322
912			\|a GBV_ILN_4323
912			\|a GBV_ILN_4324
912			\|a GBV_ILN_4325
912			\|a GBV_ILN_4326
912			\|a GBV_ILN_4333
912			\|a GBV_ILN_4335
912			\|a GBV_ILN_4336
912			\|a GBV_ILN_4338
912			\|a GBV_ILN_4346
912			\|a GBV_ILN_4367
912			\|a GBV_ILN_4392
912			\|a GBV_ILN_4393
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author_variant	f m fm h l hl x w xw g q gq b o bo d s ds
matchkey_str	article:20491891:2015----::piiepoutootaseibfaomroadfsrnbct
hierarchy_sort_str	2015
publishDate	2015
allfields	10.1186/s40104-015-0044-x doi (DE-627)SPR032858035 (SPR)s40104-015-0044-x-e DE-627 ger DE-627 rakwb eng 630 ASE Meng, Fanli verfasserin aut Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method. Buffalo (dpeaa)DE-He213 Cytoplasmic injection (dpeaa)DE-He213 Plasmid (dpeaa)DE-He213 Transgenic (dpeaa)DE-He213 Zygote (dpeaa)DE-He213 Li, Hui verfasserin aut Wang, Xiaoli verfasserin aut Qin, Guangsheng verfasserin aut Oback, Björn verfasserin aut Shi, Deshun verfasserin aut Enthalten in Journal of animal science and biotechnology Beijing, 2010 6(2015), 1 vom: 20. Okt. (DE-627)669003441 (DE-600)2630162-3 2049-1891 nnns volume:6 year:2015 number:1 day:20 month:10 https://dx.doi.org/10.1186/s40104-015-0044-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_121 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_647 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2036 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2700 GBV_ILN_2817 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4367 GBV_ILN_4392 GBV_ILN_4393 GBV_ILN_4700 GBV_ILN_4753 AR 6 2015 1 20 10
spelling	10.1186/s40104-015-0044-x doi (DE-627)SPR032858035 (SPR)s40104-015-0044-x-e DE-627 ger DE-627 rakwb eng 630 ASE Meng, Fanli verfasserin aut Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method. Buffalo (dpeaa)DE-He213 Cytoplasmic injection (dpeaa)DE-He213 Plasmid (dpeaa)DE-He213 Transgenic (dpeaa)DE-He213 Zygote (dpeaa)DE-He213 Li, Hui verfasserin aut Wang, Xiaoli verfasserin aut Qin, Guangsheng verfasserin aut Oback, Björn verfasserin aut Shi, Deshun verfasserin aut Enthalten in Journal of animal science and biotechnology Beijing, 2010 6(2015), 1 vom: 20. Okt. (DE-627)669003441 (DE-600)2630162-3 2049-1891 nnns volume:6 year:2015 number:1 day:20 month:10 https://dx.doi.org/10.1186/s40104-015-0044-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_121 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_647 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2036 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2700 GBV_ILN_2817 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4367 GBV_ILN_4392 GBV_ILN_4393 GBV_ILN_4700 GBV_ILN_4753 AR 6 2015 1 20 10
allfields_unstemmed	10.1186/s40104-015-0044-x doi (DE-627)SPR032858035 (SPR)s40104-015-0044-x-e DE-627 ger DE-627 rakwb eng 630 ASE Meng, Fanli verfasserin aut Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method. Buffalo (dpeaa)DE-He213 Cytoplasmic injection (dpeaa)DE-He213 Plasmid (dpeaa)DE-He213 Transgenic (dpeaa)DE-He213 Zygote (dpeaa)DE-He213 Li, Hui verfasserin aut Wang, Xiaoli verfasserin aut Qin, Guangsheng verfasserin aut Oback, Björn verfasserin aut Shi, Deshun verfasserin aut Enthalten in Journal of animal science and biotechnology Beijing, 2010 6(2015), 1 vom: 20. Okt. (DE-627)669003441 (DE-600)2630162-3 2049-1891 nnns volume:6 year:2015 number:1 day:20 month:10 https://dx.doi.org/10.1186/s40104-015-0044-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_121 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_647 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2036 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2700 GBV_ILN_2817 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4367 GBV_ILN_4392 GBV_ILN_4393 GBV_ILN_4700 GBV_ILN_4753 AR 6 2015 1 20 10
allfieldsGer	10.1186/s40104-015-0044-x doi (DE-627)SPR032858035 (SPR)s40104-015-0044-x-e DE-627 ger DE-627 rakwb eng 630 ASE Meng, Fanli verfasserin aut Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method. Buffalo (dpeaa)DE-He213 Cytoplasmic injection (dpeaa)DE-He213 Plasmid (dpeaa)DE-He213 Transgenic (dpeaa)DE-He213 Zygote (dpeaa)DE-He213 Li, Hui verfasserin aut Wang, Xiaoli verfasserin aut Qin, Guangsheng verfasserin aut Oback, Björn verfasserin aut Shi, Deshun verfasserin aut Enthalten in Journal of animal science and biotechnology Beijing, 2010 6(2015), 1 vom: 20. Okt. (DE-627)669003441 (DE-600)2630162-3 2049-1891 nnns volume:6 year:2015 number:1 day:20 month:10 https://dx.doi.org/10.1186/s40104-015-0044-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_121 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_647 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2036 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2700 GBV_ILN_2817 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4367 GBV_ILN_4392 GBV_ILN_4393 GBV_ILN_4700 GBV_ILN_4753 AR 6 2015 1 20 10
allfieldsSound	10.1186/s40104-015-0044-x doi (DE-627)SPR032858035 (SPR)s40104-015-0044-x-e DE-627 ger DE-627 rakwb eng 630 ASE Meng, Fanli verfasserin aut Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method. Buffalo (dpeaa)DE-He213 Cytoplasmic injection (dpeaa)DE-He213 Plasmid (dpeaa)DE-He213 Transgenic (dpeaa)DE-He213 Zygote (dpeaa)DE-He213 Li, Hui verfasserin aut Wang, Xiaoli verfasserin aut Qin, Guangsheng verfasserin aut Oback, Björn verfasserin aut Shi, Deshun verfasserin aut Enthalten in Journal of animal science and biotechnology Beijing, 2010 6(2015), 1 vom: 20. Okt. (DE-627)669003441 (DE-600)2630162-3 2049-1891 nnns volume:6 year:2015 number:1 day:20 month:10 https://dx.doi.org/10.1186/s40104-015-0044-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_121 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_647 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2036 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2190 GBV_ILN_2336 GBV_ILN_2470 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2700 GBV_ILN_2817 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4367 GBV_ILN_4392 GBV_ILN_4393 GBV_ILN_4700 GBV_ILN_4753 AR 6 2015 1 20 10
language	English
source	Enthalten in Journal of animal science and biotechnology 6(2015), 1 vom: 20. Okt. volume:6 year:2015 number:1 day:20 month:10
sourceStr	Enthalten in Journal of animal science and biotechnology 6(2015), 1 vom: 20. Okt. volume:6 year:2015 number:1 day:20 month:10
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Buffalo Cytoplasmic injection Plasmid Transgenic Zygote
dewey-raw	630
isfreeaccess_bool	true
container_title	Journal of animal science and biotechnology
authorswithroles_txt_mv	Meng, Fanli @@aut@@ Li, Hui @@aut@@ Wang, Xiaoli @@aut@@ Qin, Guangsheng @@aut@@ Oback, Björn @@aut@@ Shi, Deshun @@aut@@
publishDateDaySort_date	2015-10-20T00:00:00Z
hierarchy_top_id	669003441
dewey-sort	3630
id	SPR032858035
language_de	englisch
fullrecord	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">SPR032858035</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230519071821.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">201007s2015 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s40104-015-0044-x</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)SPR032858035</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(SPR)s40104-015-0044-x-e</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="082" ind1="0" ind2="4"><subfield code="a">630</subfield><subfield code="q">ASE</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Meng, Fanli</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2015</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. 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author	Meng, Fanli
spellingShingle	Meng, Fanli ddc 630 misc Buffalo misc Cytoplasmic injection misc Plasmid misc Transgenic misc Zygote Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
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topic_title	630 ASE Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection Buffalo (dpeaa)DE-He213 Cytoplasmic injection (dpeaa)DE-He213 Plasmid (dpeaa)DE-He213 Transgenic (dpeaa)DE-He213 Zygote (dpeaa)DE-He213
topic	ddc 630 misc Buffalo misc Cytoplasmic injection misc Plasmid misc Transgenic misc Zygote
topic_unstemmed	ddc 630 misc Buffalo misc Cytoplasmic injection misc Plasmid misc Transgenic misc Zygote
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title	Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
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title_full	Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
author_sort	Meng, Fanli
journal	Journal of animal science and biotechnology
journalStr	Journal of animal science and biotechnology
lang_code	eng
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author_browse	Meng, Fanli Li, Hui Wang, Xiaoli Qin, Guangsheng Oback, Björn Shi, Deshun
container_volume	6
class	630 ASE
format_se	Elektronische Aufsätze
author-letter	Meng, Fanli
doi_str_mv	10.1186/s40104-015-0044-x
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title_sort	optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
title_auth	Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
abstract	Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.
abstractGer	Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.
abstract_unstemmed	Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.
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title_short	Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection
url	https://dx.doi.org/10.1186/s40104-015-0044-x
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author2	Li, Hui Wang, Xiaoli Qin, Guangsheng Oback, Björn Shi, Deshun
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up_date	2024-07-03T15:08:59.547Z
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fullrecord_marcxml	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">SPR032858035</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230519071821.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">201007s2015 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s40104-015-0044-x</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)SPR032858035</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(SPR)s40104-015-0044-x-e</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="082" ind1="0" ind2="4"><subfield code="a">630</subfield><subfield code="q">ASE</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Meng, Fanli</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2015</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i) how much linear DNA to inject and ii) when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein (EGFP) plasmid into buffalo zygotes. Results First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/μL DNA was significantly higher than from those injected with 5 μg/mL. However, 50 ng/μL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/μL DNA. Second, zygotes injected early (7–8 h post-insemination [hpi]) developed better than those injected at mid (12–13 hpi) or late (18–19 hpi) time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections (16.4 % vs 7.7 %). With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Buffalo</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Cytoplasmic injection</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Plasmid</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Transgenic</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Zygote</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Li, Hui</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Wang, Xiaoli</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Qin, Guangsheng</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Oback, Björn</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Shi, Deshun</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Journal of animal science and biotechnology</subfield><subfield code="d">Beijing, 2010</subfield><subfield code="g">6(2015), 1 vom: 20. 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Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

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