Cytoplasmic Injection of Circular Plasmids Allows Targeted Expression in Mammalian Embryos

Abstract

Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter-controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…As the developmental competence of porcine zygotes is not or only minimally affected by the cytoplasmic injection, it is sufficient to transfer about 30 embryos per recipient sow, thus allowing for the reduction in the number of animals used. Cytoplasmic injection avoids high-speed centrifugation of opaque zygotes 5,54 , which is necessary when using pronuclear injection, and it avoids invasive removal of metaphase plates from oocytes, which is an essential step of porcine SCNT. The majority of transgene integration events represent specific transpositions of monomeric transposon units (Supplementary Fig.…”

“…2a, b) is a relatively gentle injection method that avoids high-speed centrifugation of porcine zygotes and invasive removal of metaphase plates from porcine oocytes, which is an essential step of SCNT. The developmental competence of mammalian zygotes is not or only minimally affected by CPI 54,55 , thus allowing for the reduction of animal numbers used. The protocol consists of the following major parts:…”

“…These steps include superovulation of sows and collection of zygotes, microinjection of the plasmid mixture into zygotes, surgical embryo transfer to recipient sows followed by after-care of recipient animals and ultrasound scanning for implantations. To set up the CPI method, it may be helpful to start with in vitro culture of microinjected embryos to allow the assessment of ratios of successful injections and viable embryos 54 . In vitro culture for 5 days should lead to development of blastocyst stages, consisting of an inner cell mass, the trophoblast and a central, fluid-filled cavity 54 .…”

“…To set up the CPI method, it may be helpful to start with in vitro culture of microinjected embryos to allow the assessment of ratios of successful injections and viable embryos 54 . In vitro culture for 5 days should lead to development of blastocyst stages, consisting of an inner cell mass, the trophoblast and a central, fluid-filled cavity 54 . The Venus transposon allows a direct assessment of successfully injected embryos by fluorescence microscopy.…”

“…but alone no proof for SB-catalyzed transposition, as episomal plasmids will also be transcribed 54,55 .…”