Electroporation and in vitro culture of early rabbit embryos

The functional interrogation of factors underlying early mammalian development is necessary for the understanding and amelioration of human health conditions. The associated article [1] reports on the molecular characterization of markers of neural crest cells in gastrula and neurula stage rabbit embryos. This article presents survival data of rabbit embryos cultured in vitro, as well as immunofluorescence data for molecular markers of neural crest cells following approximately 24-h of culture. Lastly, towards the functional analysis of early neural crest and other developmental genes, this article provides data on the introduction of exogenous DNA into early stage rabbit embryos using electroporation.


a b s t r a c t
The functional interrogation of factors underlying early mammalian development is necessary for the understanding and amelioration of human health conditions. The associated article [1] reports on the molecular characterization of markers of neural crest cells in gastrula and neurula stage rabbit embryos. This article presents survival data of rabbit embryos cultured in vitro, as well as immunofluorescence data for molecular markers of neural crest cells following approximately 24-h of culture. Lastly, towards the functional analysis of early neural crest and other developmental genes, this article provides data on the introduction of exogenous DNA into early stage rabbit embryos using electroporation.
& 2018 Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area Developmental Biology Type of data

Value of the data
This data demonstrates the first successful incorporation of exogenous DNA into rabbit embryos using electroporation.
Embryo survival data and marker gene expression following 24-h culture at multiple stages will be relevant to inform future experiments using the rabbit model system.
Towards efforts to examine the functional role of early developmental genes in the rabbit, these data together provide important experimental guidance on embryological manipulations using the rabbit embryo.

Data
We present data on the survival rates of in vitro cultured gastrula and neurula stage rabbit embryos, and use immunofluorescence to stain cultured rabbit embryos for molecular markers of neural crest cells, including Pax3, Pax7, and Sox10. We also provide data on the cellular introduction of exogenous DNA (morpholino oligonucleotides and plasmids) into rabbit embryos using electroporation, and the successful culture thereafter.

Embryo collection and staging
Timed-pregnant New Zealand White rabbits (Millbrook Breeding Labs, Amherst, MA) were euthanized, and uteri removed and stored in 1x PBS at 4°C. Embryos were dissected from the uterine tissue in sterile 1x PBS containing 5% Fetal Bovine Serum (FBS) and 100 U/mL Penicillin-Streptomycin (Thermo Fisher). Embryos were staged as previously reported, with pre-gastrula embryos according to Viebahn et al. [2,3], and gastrula and neurula embryos according to chick developmental stages [3,4]. Rabbit embryos were obtained and experiments performed at Yale University, following the approval and guidelines of IACUC.

in vitro culture of rabbit embryos
Whole rabbit embryos between St. 1 and 3 somite stage were cultured using the "ring method" as previously described [5] at 37°C with 5% CO2 for 19-22. Following culture, embryos were cryosectioned and processed for immunofluorescence as described below. Successful cultures were marked as those embryos demonstrating normal morphology along the entire rostral-caudal axis (Table 1).

Immunohistochemistry
For immunohistochemistry, rabbit embryos were fixed in 4% paraformaldehyde for 30 min to 1.5 h (depending on the stage) at room temperature. Embryos were embedded in gelatin and cryosectioned (Leica CM1900), or subjected to whole mount immunofluorescence. Sections were stained for markers of NC including AP2α, Msx1/2, Pax3, Pax7, Sox9, and Sox10, and the neural markers Sox2/3, as described in the associated article [1].

Electroporation of rabbit embryos
Embryos (St. 4 þ to St. 6 and 3-4ss) intended for electroporations were prepared for whole mount culture as above. Following the removal of excess culture media, embryos were injected with a mixture of the following: Sucrose (1.7-2%), non-targeting Lissamine-morpholino oligonucleotide (0.33-0.4 mM), pCIG (1ug/uL), and Fast Green. Injections typically targeted the right side of the embryonic disc. Electroporations were performed with 5.5-6 V (n¼4; St. 4 þ/5) or 6.5 V (n ¼7; St. 5/6 to 3-4ss) with 5 pulses of 50 ms "On," 100 ms "Off." Rings were placed over the extraembryonic tissue framing the embryos post-electroporation, and subsequently filled with culture media. Electroporated embryos were cultured overnight. All embryos electroporated at 5.5-6 V demonstrated normal morphology post-culture, and morpholino signal was observed in all examples (n ¼4/4). All electroporations were performed with a dual-pronged rod, as has previously been described for the in vivo electroporation of chick embryos [6]. We also note that all embryos electroporated with 6.5 V After culture, the 7ss embryos displays normal morphology including formation of neural folds, somites, and heart primordia.

Table 1
Survival rates for the in vitro culture of rabbit embryos. All embryos were cultured using the "ring culture" method as described by Püschel et al. [5]. Rabbit embryos can be cultured from St. 1 to St. 2 with a high degree of success (67%); however, in our hands, the success rate drops dramatically when embryos between St. 3 and St. 4 are placed into culture (17%). Following gastrulation stages, rabbit embryos at St. 5 and older can be cultured up to the 7-8ss, with embryos generally demonstrating normal morphology (i.e. neural folds/neural tube and somites) (Fig. 1C-D). The success rate for these older cultures hovers between 67-83%, depending on the starting embryonic stage. We also note the successful culture of a St. 3 embryo cultured to the St. 7 cranial stage (following 48-h culture), although this embryo was deformed caudally and not included in the table.
Although the successful culture of rabbit embryos as young as St. 4 through the early somite stages has been reported, these embryos often display abnormal marker gene expression [7]. , with these embryos retaining morpholino expression on the side of electroporation ( Fig. 2 and 3).  We also observed a small number of GFP-expressing cells in embryos co-injected with lissamine-MO and pCIG (G, H) (n ¼ 2/4). However, we note that GFP expression was observed n ¼ 2/4 embryos tested.