23 Matching Annotations
  1. Mar 2021
    1. Such cloning al-lows the production of genetically uniform monkeys as animalmodels for basic research in primate biology and for studying hu-man disease mechanisms and therapeutic treatments

      future directions

    2. ccess of cloning of a non-human primate bySCNT to the optimization of the nuclear transfer protocol, theuse of fetal cell nuclei, and epigenetic modifications byKdm4dand TSA, and all of them together greatly improved the qualityof blastocyst development and pregnancy rate.


    3. Taken together, the expression of H3K9me3demethylaseKdm4dsignificantly improved epigenetic reprog-ramming in monkey SCNT embryos similar to that found in otherspecies


    4. Dppa2,Dppa4, andMycwere repressed by H3K9me3in monkey SCNT embryos

      unintended genes that were repressed by the methylation that could have been responsible for the poor development of SCNT monkey embryos

    5. RNA-seq


    6. We found that a much higher percentage(17/38, or 44.7%) ofKdm4dmRNA-injected SCNT embryosderived from fetal monkey fibroblasts were able to develop intoFigure 1. Procedure for Monkey SCNTUsing Fetal Monkey Fibroblasts(A) Fetal monkey fibroblasts in primary culture.(B and C) Spindle-chromosome complex (arrow-head) in a monkey MII oocyte before (B) and after(C) removal.(D–F) An oocyte with a slit in the zona pellucida (D,arrowhead) induced by laser irradiation, and theHVJ-E-incubated fibroblast (arrowhead) before (E)and after (F) injection into the perivitelline space.(G) Spindle-like structure formed by the donorfibroblast nucleus after fibroblast-cytoplast fusion.(H) Single nucleus formed after embryo activationwith ionomycin and DMAP.(I) Blastocysts developed fromKdm4dmRNA-injected embryos, which were produced by SCNTusing fetal fibroblasts (arrowheads: ICM).(J–L) Example images of blastocysts with andwithout normal development of ICM shown at ahigher resolution. Blastocysts with a prominentICM (arrowhead) obtained by SCNT (J), blasto-cysts with a prominent ICM (arrowhead) obtainedby ICSI (K), and poor-quality blastocysts withoutclear ICM obtained by SCNT (L).All scale bars, 60mm.blastocysts, among which a large fraction(11/17, or 64.7%) showed prominent ICMsimilar to the ICM in embryos obtained byintracytoplasmic sperm injection (Figures1I–1L,2A, and 2B). We also tested theeffect ofKdm4dmRNA injection onSCNT embryos derived from cumuluscells of adult female monkeys (from which the oocytes wereobtained) and found that all SCNT embryos showed a singlepronucleus after activation under I/D/T condition (Figures S2A–S2D). The majority of them (24/33, or 72.7%) developed intoblastocysts, most of which (15/24, or 62.5%) showed prominentICM. By contrast, in the absence ofKdm4dmRNA injection, only5% (1/20) of SCNT embryos derived from cumulus cellsdeveloped into blastocysts, none of which showed prominentICM
    7. Analysis of RNA-seq datasets of monkeyICSI embryos at 4- and 8-cell stage (n = 4 embryos each) ledto identification of 3,997 regions (20–160 kb) that were ex-pressed at least 5-fold higher (FC > 5) in 8-cell embryos ascompared to 4-cell embryos (Data S1), indicating massive up-regulation of genes during early embryonic development.
    8. Kdm4d

      a human enzyme - why was this added?

    9. Clear spindle-like structure was detected after thefusion of the fetal monkey fibroblast with the enucleated oocyte(Figures 1G andS1A). About 1–2 hr after the fusion, ‘‘recon-structed’’ oocytes were activated with ionomycin and 6-dime-thylaminopurine (I/D). All activated embryos showed a singlepronucleus (Figures 1H andS1B) and were further cultured forin vitrodevelopment

      Evidence showing success with the fusion of the fibroblasts with the enucleated oocyte.

    10. laser lesion of zona pellucida

      SOS technique

    11. Hemagglutinin Virus of Japan


    12. polarized-lightimaging

      what is this?

    13. suggesting that RRRs are conservedamong mammalian species

      what does this mean?

    14. trimethylation (H3K9me3) modification


    15. earlyblastomeres

      The cells in cleavage stage embryos

    16. nuclear transfer

      The steps involve removing the DNA from an oocyte (unfertilised egg), and injecting the nucleus which contains the DNA to be cloned.

    17. previous failure of SCNTappears to be caused by inappropriate reprogramming of the so-matic nucleus for supporting the development of transplantedembryos.

      reason for past failures

    18. As species closer to humans,non-human primates are ideal animal models for studyingphysiological functions unique to primates and for developingtherapeutic treatments of human diseases (Izpisua Belmonteet al., 2015; Jennings et al., 2016). Animal models with geneticuniformity are often desirable (Schramm and Paprocki, 2004),but an inbreeding approach as used in generating rodent modelsis not practical for non-human primates

      purpose of the experiment

    19. In both cases, genetic analyses confirmedthat the nuclear DNA and mitochondria DNA of themonkey offspring originated from the nucleus donorcell and the oocyte donor monkey, respectively.Thus, cloning macaque monkeys by SCNT is feasibleusing fetal fibroblasts.

      Answers the question of whether using SCNT to clone monkeys is possible. They back up this claim using their findings from genetic analyses.

    20. trans-planted SCNT embryos in surrogate monkeys. ForSCNT using fetal monkey fibroblasts, 6 pregnancieswere confirmed in 21 surrogates and yielded2 healthy babies. For SCNT using adult monkeycumulus cells, 22 pregnancies were confirmed in42 surrogates and yielded 2 babies that were short-lived

      SCNT using fetal monkey fibroblasts vs. SCNT using adult monkey cumulus cells

    21. establish animal models forprimate biology and biomedical research

      can be used for human medical research

    22. Genetic analysis confirmed the clonal origin of the SCNTmonkey offspring

      was able to prove that the somatic cell nuclear transfer was successful in the monkey offspring

    23. cumulus cells

      Cumulus cells are defined as a group of closely associated granulosa cells that surround the oocyte and participate in the processes of oocyte maturation and fertilization.