# Pluripot Stem Cells for Transgenesis in the Rabbit: A Utopia?
## 3 Rabbit Chimeras
### Colonization and Chimerism  
It is important to distinguish embryo colonization, which corresponds to the presence of donor cells in preimplantation embryos, from chimerism, which represents the participation of donor cells to embryonic development in parallel with host cells. The mere presence of donor cells in the ICM of recipient blastocyst does not presage their participation in embryonic development and may only reflect their autonomous development [94]. Thus, a chimeric fetus must be made up of a homogeneous mosaic of cells of both origins; this was the case for cynomolgus chimeras obtained with naive-like monkey PSCs, even if the rate of chimerism was still low [85]. Similar to primed primate PSCs, primed rbESCs were unable to colonize the ICM of host blastocysts after microinjection at the morula stage [45; 47; 61]. However, primed rbiPSCs yielded colonized blastocysts after the aggregation of morula with cells expressing LacZ or green fluorescent protein (GFP) marker genes [47] (Figure 2). Similarly, rbESCs derived in the presence of serum and in the absence of FGF2 produced early blastocysts with GFP-positive cells in the ICM, with 2% or 16% colonized embryos, respectively, depending on the lineage [51]. These percentages are comparable to those obtained with reprogrammed rbiPSCs cultured in 2 iLIF medium containing KOSR, forskolin, and kenpaullone [72]. In vitro, reprogrammed rbEKA cells allowed a higher rate of colonized embryos to be obtained, up to 33% at the E3.5 stage and 25% at the E4.5 stage, but the percentage of embryos with microinjected cells into the ICM was only 10% on average [65]. In the same study, after the transfer of rbEKA-treated embryos into surrogate females, six E6-stage pre-gastrula (1.4%) and two E10-stage fetuses (3%) were found colonized by rbEKA cells [65] (Figure 2). The number and the localization of GFP-expressing rbEKA cells showed the amplification and the participation of the injected cells to the formation of the host epiblast. Pre-gastrula showed a fairly uniform distribution of fluorescent cells throughout the embryonic disc and the only gastrula obtained showed GFP-positive cells in the primitive streak, and therefore potentially entering into the process of gastrulation. However, the percentage of colonized embryos decreased as embryonic development progressed, and above all, the rate of colonization continually declined. Even though most ICMs appeared to be massively colonized at the E3.5 and E4.5 stages, the rate of chimerism gradually decreased to 1% in fetuses according to a semi-quantitative PCR estimate [65]. The injected cells observed in these colonized embryos were far from being in the majority, as is the case in murine chimeras produced with a single donor cell [95]. These low colonization rates are consistent with those of interspecific chimeras generated by microinjection of naive-like reprogrammed primate PSCs into mouse or pig embryos [96; 97; 98].  
Figure 2: Embryonic colonization capacity of several rabbit pluripotent stem cell lines produced by our research group [47; 51; 65] or the Japanese team of [PERSON] [61; 72]. rbESC: rabbit embryonic stem cell; rbiPSC: rabbit induced pluripotent stem cell; KOSR: knockout serum replacement; FGF2: fibroblast growth factor 2; FBS: fetal bovine serum; LIF: leukemia inhibitory factor; 2i: MEK and GSK3\(\beta\) inhibitors; Kip: kenpaullone; Fk: forskolin; hOSKM: human OCT4, SOX2, KLF4, and c-MYC factors; CKF: clumps/KOSR/FGF2; AKSL: accutase/KOSR + FBS/LIF; rbEKA: rabbit enhanced KLF activity. Scale bar = 50 \(\upmu\)m.