|With No Help from Mother - “Generation of Genetically Modified Mice by Oocyte Injection of Androgenetic Haploid Embryonic Stem Cells”|
Haploid stem cells allow functional screening for genes involved in diverse cellular and developmental processes, and have the potential to be used in place of gametes to create animal models (Kaufman et al and Yan et al). However, mouse embryonic stem cells (ESCs) originally established from haploid embryos display a diploid karyotype (Kaufman et al), but recent studies have derived relatively stable haploid ESCs (haESCs) from mouse parthenogenetic embryos and applied them in forward and reverse genetic screens (Elling et al andLeeb and Wutz). Yet the parthenogenetic nature of the haESCs suggests that they may not support full-term development of embryos. And therefore it is still unclear whether haESCs carrying specific traits can be converted into a mouse model that produce progeny and deliver the genetic traits to the next generation. Now, in a study published in Cell, researchers from the group of Jinsong Li from the Shanghai Institutes for Biological Sciences, China have established protocols for the derivation of fully pluripotent haESCs from androgenetic (AG) blastocysts, which following injection into oocytes lead to the generation of fertile animals (Yang, Shi, Wang et al).
Two different approaches were adopted for the generation of AG haploid mouse embryos. Nuclear transfer of a haploid sperm head from an Oct4-EGFP transgenic mouse (C57BL/6 background) into an enucleated oocyte led to a 21% blastocyst development rate in vitro giving 34 ESC lines. Of these, 4 lines (AGH-OG-1 to 4) were maintained as haESCs through multiple rounds of haploid cell enrichment by FACS (fluorescence activated cell sorting). The second approach involved the removal of the female pronucleus from oocytes fertilized by Actin-EGFP transgenic male mice leading to a 17% blastocyst development rate in vitro, ultimately giving 5 ESC lines, of which one haploid ESC line (AGH-EG-1) was obtained after multiple rounds of FACS following passaging in vitro. Overall, 5 AG-haESC lines were generated which could be expanded for more than 30 passages, exhibited no Y chromosome, and were karyotypically and genomically stable. Further more, these haploid cells were shown to be fully pluripotent. Morphologically the cells resembled normal diploid mESCs, expressed Nanog, Oct4, Sox2, and SSEA1, and gene expression profile analysis demonstrated a high correlation between AG-haESCs and the diploid ESCs, but not mouse embryonic fibroblast (MEFs). Developmental analysis also demonstrated that AG-haESCs could contribute to the germline and produce chimeric mice, of which some survived to adulthood with a high degree of somatic contribution by ESCs, however, no haploid cells could be observed in vivo from embryonic day (E) 6.5 onwards although in vitro differentiation with retinoic acid treatment for 6 days showed that both undifferentiated and differentiated populations contained haploid cells suggesting that that haploidy can exist, at least transiently, in differentiating cells. As expected from the androgenetic origin of AG-haESCs, all paternally imprinted genes (maternally expressed genes) were downregulated, except for the H19 gene, and maternally imprinted genes (paternally expressed genes) were upregulated, suggesting that the AG-haESCs largely maintained a typical paternal imprinting status.
Next, the ability of AG-haESCs to support full-term development of mouse embryos upon injection into mature oocytes was analysed. Synchronisation of AG-haESCs in metaphase, which allows for increased blastocyst rate in nuclear transfer (Ono et al 2001a and Ono et al 2001b), in addition to the selection of small cells, most of which were haploid, led to reconstructed embryos containing diploid DNA. Of 19 ESC lines established from 40 blastocysts, 17 ESC lines were diploid reflecting a successful procedure. Developmental potential was examined by transferring two-cell embryos or blastocysts into oviducts or uteri of pseudopregnant females, respectively. Out of 553 transferred two-cell embryos and 424 blastocysts derived from all five AG-haESC lines (passage 7 to passage 22), a total of 46 live pups were recovered by caesarean section at 19.5 days of gestation, all of which were female and carried an EGFP transgene that originated from the AG-haESCs. The mice, termed semi-cloned (SC) by the authors, were either normal with a typical newborn body weight or developmentally retarded. The developmentally retarded mice all died within an hour of birth and had an unmethylated H19 locus, compared to live newborn mice, which had a methylated H19 locus. Furthermore, growth-retarded pups exhibited significantly lower Igf2 expression in major organs than control mice. Of the live born mice (18), 14 grew to adulthood and dissection of one animal demonstrated the appearance of EGFP in the ovaries and germinal vesicle (GV) oocytes. The mating of one mouse led to the birth of 16 pups, of which 50% were EGFP positive and were both male and female indicating that female SC mice derived from AG-haESCs are capable of normal gametogenesis.
Next the ability of specific genetic alterations to be introduced into these haESCs through homologous recombination was examined, using the introduction of a PGK-neo drug selection cassette into the locus of a gene (Vwce, a gene encoding ‘‘vonWillebrand factor C and EGF domains’’-containing protein presumably involved in the Wnt-signaling pathway) chosen for its likeliness to avoid effects on cellular function. Following selection with both G418 and Ganciclovir, 90 double-positive colonies were found, of which 12 clones contained the targeted allele only. 4 of these clones were established as lines through consecutive passaging and FACS sorting for haploid cells and of 444 embryos constructed with these cells, one live-birth SC pup was generated. Although the pup carried the targeted allele, it was growth retarded and died shortly after birth, which was attributed to loss of imprinting and the relatively late passage of the targeted AG-haESCs (passage 28). This overall suggests that AG-haESCs are amenable to standard gene targeting manipulation, and in principle, can allow for the generation of genetically modified animals.
Taken together, these results demonstrate that haESCs derived from androgenetic blastocysts are pluripotent as normal diploid ESCs and when injected into oocytes, can introduce genetic traits into the resulting mice which can be further transmitted to offspring. This therefore suggests that haESCs can indeed be converted into mouse models; a prerequisite for extending genetic analysis to the organism level. However, failures in imprinting leading to the production of growth retarded mice suggests that the appropriate derivation and culture conditions still require to be uncovered to allow the stable maintenance of imprinting marks in AG-haESCs, which would facilitate the potential applications of these cells.
Elling, U., et al. (2011).
Kaufman, M.H., et al (1983).
Leeb, M., and Wutz, A. (2011).
Ono, Y., et al (2001a).
Ono, Y., et al (2001b).
Yan, H., et al. (2000).
Yang, H., et al.
STEM CELLS correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.
Original study from Cell Stem Cell.