Abstract
Cytoplasmic microinjection (CI) of foreign gene into in vivo fertilized zygotes has emerged as a useful tool for transgenic pig production. In the current study, we investigated factors affecting transgenic efficiency and developmental potential of parthenogenetic (PA) and in vitro-fertilized (IVF) porcine embryos produced by CI. These factors included adding of RNase inhibitor, DNA or RNA concentration, injection time, and different structures of plasmids. Our results showed that adding of 1–4 U/μL of RNase inhibitor did not have negative effect on development potential of CI-PA embryos, and RNase inhibitor injection significantly increased EGFP expressing rate of CI-PA embryos. High injection DNA concentration and long injection interval after PA significantly reduced blastocyst formation. Different molecular structures such as DNA or RNA affected CI-PA embryos development, and RNA had little harmful effect on pig’s early embryonic development. EGFP expression rate of CI-IVF embryos was improved following the increase of foreign DNA concentration, but blastocyst formation rate was decreased. Injection time after IVF did not show any significant difference on embryonic development, but longer interval resulted in a significantly lower EGFP expressing rate. Cas9 mRNA and myostatin (GDF-8) sgRNA co-injection indicated that the mutation rate of CI-IVF group was significantly higher than that of CI-PA. The CI-IVF-generated embryos were then transferred to six recipient pigs, but no live piglets were obtained. The following pronuclear formation assessment showed more than 76.1% IVF zygotes were polyspermy. These results demonstrate that CI-PA and CI-IVF were effective methods for production of transgenic pig embryos. However, polyspermic fertilization and poor quality of porcine IVF blastocysts are still the main problem of resulting in pregnancy failure.
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This work was funded by the Hebei Provincial National Natural Science Fund (H2016314002) and the China 863 high-tec Project (2011AA100607).
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Editor: Tetsuji Okamoto
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Fig. 1
Transgene vector constructions. pcDNA-EGFP-N1 (GIF 7 kb)
PRL-EGFP: EGFP Expression Vector with PRL ORF Controled by BCN promoter (GIF 11 kb)
TALEN-BMP15 and TALEN- FoxO3: The TALEN expression plasmid for knocking-out BMP15 and FoxO3 of porcine (GIF 9 kb)
hSpCas9 expression plasmid with a NLS at the N-terminal (GIF 9 kb)
Cas-BMP15, Cas-FoxO3 and Cas-GDF8 gRNA expression vector with a PsiI restriction endonuclease site for linearized (GIF 8 kb)
Fig. 2
Sequence alignment of GDF8 IVF mutant embryos (GIF 187 kb)
(GIF 244 kb)
(GIF 231 kb)
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Liu, S., Liu, X., Huang, H. et al. Factors affecting efficiency of introducing foreign DNA and RNA into parthenogenetic or in vitro-fertilized porcine eggs by cytoplasmic microinjection. In Vitro Cell.Dev.Biol.-Animal 52, 713–722 (2016). https://doi.org/10.1007/s11626-016-0025-1
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DOI: https://doi.org/10.1007/s11626-016-0025-1