Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs

Abstract

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed "nuclear injection". To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

“…The in vitro embryos were produced as previously described (41,42). In brief, antral follicles on the surface of ovaries obtained from a local abattoir with a size of 3-6 mm in diameter were aspirated for the collection of cumulus-oocyte complexes (COCs) (42).…”

“…During the rst 22 hours, the COCs were cultured in maturation medium supplemented with human chorionic gonadotropin and pregnant mare serum gonadotropin, followed by 22-24 hours of culture in hormone free maturation medium in a humidi ed atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5°C (42). The in vitro fertilization was performed using frozen sperm derived from the same Duroc boar as used for the in vivo developed embryos to reduce an in uence on genetic variation (41). A group of 20 matured oocytes was co-incubated for 7 hours with 1.0 × 10 6 cells/mL in a porcine fertilization medium (Functional PeptideCo., Yamagata, Japan) in a humidi ed atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5°C (41).…”

“…The in vitro fertilization was performed using frozen sperm derived from the same Duroc boar as used for the in vivo developed embryos to reduce an in uence on genetic variation (41). A group of 20 matured oocytes was co-incubated for 7 hours with 1.0 × 10 6 cells/mL in a porcine fertilization medium (Functional PeptideCo., Yamagata, Japan) in a humidi ed atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5°C (41). After fertilization, the cumulus cells and excess sperm were removed from the presumed zygotes and were cultured in Porcine Zygote medium-5 (Functional Peptide Co., Yamagata, Japan) in a humidi ed atmosphere of 5% CO 2 , 5% O 2 , and 90% N 2 at 38.5°C (41).…”

Cellular and Molecular Probing of Intact Transparent Human Organs

“…Recent developments in gene editing with CRISPR/ Cas9 technology have enabled easy generation of large transgenic reporter mammals, expressing fluorescent proteins in the tissues of interest [35,36]. Therefore, we applied SHANEL clearing to INS-EGFP transgenic pig pancreas exhibiting porcine insulin gene (INS) promotor driven beta cell specific enhanced green fluorescent protein (EGFP) expression in the islets of Langerhans [37] (Fig.…”

Osteogenic and Fluorescent Characterization of Pig-Derived Mesenchymal Stem Cells Between eGFP Transgenesis and Non-viral Plasmid Transfection

“…The CAG hybrid promoter-driven eGFP-MSCs derived from the transgenic pigs have been proven to express homogeneous surface epitopes and possess classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages with stable, robust EGFP expression in all differentiated progeny. (10) However, demanding techniques such as pronuclear microinjection, expensive breeding and equipment have limited the popularity of eGFP MSCs from transgenic pigs (11,12).…”