Halley-Stott RP et al. (2010), Mammalian nuclear transplantation to Germinal V...

XB-ART-41084

Methods 2010 May 01;511:56-65. doi: 10.1016/j.ymeth.2010.01.035.

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Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.

Halley-Stott RP , Pasque V , Astrand C , Miyamoto K , Simeoni I , Jullien J , Gurdon JB .

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Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.

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Species referenced: Xenopus laevis
Genes referenced: jun kcnma1 pou5f3 sox2 vegt

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	Fig. 1. Chemical defolliculation of oocytes completely removes the oocyte follicular cells, illustrated here by Hoechst (UV) staining of follicular cell nuclei, before and after Liberase treatment.
	Fig. 2. Typical needles used by members of our group (A). The ‘rough’ breaks are achieved by ‘nipping’ the needle tip with forceps. The needle can then be further sharpened by grinding or pulling on a micro forge, to give the ‘ground’ or ‘forged’ needle tips. GV targeting can be enhanced by marking the needles at 150–250 μm and 350–450 μm giving target depth marks on the needle shaft. (B) Diagram showing needle position of ‘minimum’ (black) and ‘maximum’ (red) depth marks on a needle in relation to a correctly targeted GV.
	Fig. 3. Nuclear transfer to Xenopus oocyte GVs. Cultured cells (a) are harvested, washed and permeabilised with SLO. Successful cytoplasmic membrane permeabilisation is detected by incorporation of Trypan Blue and cytoplasmic expansion (b, b′), whereas unpermeabilised cells do not incorporate the dye (a′). SLO permeabilised cells are directly transferred into the GV of stage V and VI Xenopus oocytes (c). Oocytes are held at a 45° angle using forceps, and nuclei are transferred directly by inserting the glass nuclear transfer pipette into the centre of the animal pole of the oocyte, until it is judged to have reached the inner part of the GV (c′). Oocytes are incubated in MBS medium, supplemented with 0.1% BSA, at 16–18 °C following transplantation (e). Successful targeting to the GV is revealed after oocyte dissection and Hoechst staining (in blue, d′). (e′) It shows an isolated GV containing injected nuclei stained with Hoechst (blue).
	Fig. 4. Schematic diagram of optimized RNA extraction, reverse transcription and qPCR, illustrating the conditions used for detecting transcripts following a nuclear transplant experiment (a). RNA is shown in red and cDNA in black. Critical parameters and output ranges are shown in the notes below the figures. Starting RNA quantities of a typical experimental series can be recovered from oocyte material in linearly proportional amounts following RNA extraction (b). Conversion of RNA to cDNA is linear across different input RNA amounts in an oocyte RNA background (c).
	Fig. 5. Reprogramming signal strength and efficiency are optimal between 100 and 300 nuclei. Transcription of Sox2 from different numbers of C2C12 nuclei injected into Xenopus oocytes and maintained at 16 °C for 48 h (a). Efficiency (absolute number of transcripts/transplanted nucleus) of Sox2 reactivation from different numbers of C2C12 nuclei injected into Xenopus oocytes and cultured at 16 °C for 48 h (b). P < 0.02, error bars are mean SD.
	Fig. 6. Accumulation of G3PDH transcripts over 4 days from oocytes injected with 10T1/2 or Thymus nuclei (a). Expression of G3PDH per injected C2C12 nucleus from different numbers of nuclei injected into Xenopus oocytes and maintained at 16 °C for 48 h (b). P < 0.02, except samples marked ∗P < 0.1, n = 3 (except first time point in A, where n = 1), error bars are mean SD.
	Fig. 7. Real Time PCR Ct values for L8, VegT and an RNA “spike” gene for nine samples of four oocytes each. Numbers adjacent to bars indicate the ratio of the ‘spike’ Ct to mean L8 and VegT Ct values.
	Fig. 8. Transcription of Nanog (a), Oct4 (b), Sox2 (c) and c-Jun (d) initiates and increases over time following nuclear transplantation into the oocyte GV for each of the four donor nuclei types examined (see key). Samples were frozen at the indicated time points following transplantation in batches of 4 oocytes and examined for transcription of the genes by qRT-PCR. Error bars represent standard deviation of mean biological replicates (n is indicated in brackets for each cell-type in the key). Δ indicates one or more replicates discarded due to GV mis-targetting for that sample group. P < 0.5, unless marked ∗0.1 > P > 0.05 or ∗∗P > 0.1.
	Fig. 9. Expression of Nanog (a), Oct4 (b), Sox2 (c) and c-Jun (d) from oocytes injected with either ES or retinoic acid differentiated ES (ESRA) nuclei (see key). (e) Transcript values for Nanog, Oct4 and Sox2 in wholes ESRA cells as compared to whole ES cells prior to permeabilization. This illustrates the typical differences between ES and 4 day retinoic acid-treated ESRA cells. Error bars represent standard deviation of mean biological replicates (n is indicated in brackets for each cell-type in the key). Δ indicates one or more replicate discarded due to GV mis-targetting for that sample group. P < 0.5, unless marked ∗0.1 > P > 0.05 or ∗∗P > 0.1.

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