Chan AP and Gurdon JB (1996), Nuclear transplantation from stably transfected...

XB-ART-18360

Int J Dev Biol 1996 Apr 01;402:441-51.

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Nuclear transplantation from stably transfected cultured cells of Xenopus.

Chan AP , Gurdon JB .

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By nuclear transplantation we have generated embryos from enucleated Xenopus eggs and nuclei of stably transfected Xenopus cell lines. We have devised a novel method of transplantation in which cell permeabilization is controlled by a temperature effect on streptolysin O-treated cells. This method is easier and quicker to operate than the conventional cell rupture technique. Single nuclei from cell lines transfected with the lacZ reporter gene were transplanted to Xenopus eggs in which the egg nuclei were destroyed by UV irradiation. We show that the lacZ transgene is transmitted from donor cells to nuclear transplant embryos. Expression of the lacZ transgene has been controlled by the elongation factor 1-alpha promoter (Krieg et al., Dev. Biol. 133: 93-100, 1989). In the nuclear transplant embryos, beta-galactosidase transcripts are expressed at the expected time of development, that is after the mid-blastula transition. In addition, we show that early embryo-specific genes, not expressed in cultured cells, are normally activated in nuclear transplant embryos. Therefore, expression of these genes can be used to monitor the effects of transfected test genes. Although most of the nuclear transplant embryos do not develop beyond the gastrula stage, explants of equatorial tissue from these embryos can undergo differentiation characterized by the expression of muscle and notochord markers. The use of nuclear transplantation, as described here, provides a means of avoiding the mosaic expression of DNA or mRNA injected into Xenopus eggs.

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Species referenced: Xenopus
Genes referenced: gsc isyna1 tbx2 tbxt
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	Fig. 1. Structure of the Xenopus ptkneo-EF1lacZ plasmid construct. Expression of the lacZ gene is controlled by rhe 4.5 kb S' lIpstream sequence of the Xenopus elongation factor 1-a gene {Krieget al.. 1989}. The 4.5 kb fragment includes the S'-untranslated region (+ 1 to +37) of rhe EF-Ja gene. The neomycin resistance gene (neaR) is driven by the HSV thymidine kinase (tk) promoter. Plasmid DNA was transfected into Xenopus XL177 cell line by electroporation. The upper panel shows the design of the antisense lacZ probe for RNase protection assay. Probes protected by !acZ transcriprs give rise to a band of362 bp
	Fig. 2. Nuclear transplant embryos at gastrula stages. (A! Nuclear transplant embryos derived from lacl expressing cell line. (B) Close up of two gastrula stage nuclear transplants. The embryos show normal blastopore /ips and yolk plugs.
	Fig. 3. Transmission of the lacZ transgene from single transplanted nuclei to nuclear transplants. A PCR-detection assay was performed on DNA extracted from single nuclear transplant embryos. The PCR products were labeled wirh 1 ).lCi la-32PldATP to enhance the sensiriviry of detection. Blastula stage nuclear transplanrs derived from (A) lacZ expressing cell lines 2. 1 and 2.14. or (8) dissociated cells from a first generation nuclear transplant; both showed a strong lacZ signal in the PCR assay. The presence of the lacZ signa! in nuclear transplants indicates stable transmission of the lacZ transgene from donor nuclei to nuclear transplant embryos. Primers specific to the endogenous EF-la gene served as internal controls for the PCR reaction
	Fig. 4. Expression of a JacZ transgene in nuclear transplants. An RNase protection assay was performed on gasrrula stage nuclear transplants derived from the laeZ expressing cell line 2.14. Transplant numbers ,. 2. 3 and 4 were harvested at stage 10.25 and numbers5. 6 and 7 at stage 10.5. All samples gave a protected fragment of expected size (362 bp) indicating rhe presence of {aeZ transcripts rather than DNA. FGF receptor was used as a loading control. The pBR322 Hinf I size marker onlyappliesto the upperpanel.
	Fig. 5. In situ hybridization of nuclear transplant embryos for lacZ expression. In situ hybridization was performed on sections of control embryos (AJ and nuclear transplants derived from cell/;ne 2.14 IBI by using a digoxygenin-fabeled anti-sense {aeZprobe. The level of the facZ rransgene expression is variable among the positively.srained cells. (CI Enlarged view of a separate section to show the perinuclear location of laeZ mRNA The same section is shown under both normal illumination and UV (D) to reveal nuclei stained with Hoechst.
	Fig. 6. Reprogramming of gene expression in nuclear transplants. RNase protection assays were performed on the same batch of RNA samples prepared from nuclear transplants as in fig 4. The RNA probes used were specific to EF-1a. Xbra, gsc. Xwnt-B and Mix. I. For gsc, Xwnt-B and Mix. 1, no transcripts were detected in unfertilized eggs or in lacZ expressing cell line 2.14 by this method. It has been shown that there is a low level of maternal Xbra transcripts in eggs (Smith et al.. 1991). Xbra expression was not detected in cell Ime 2.14 (data not shown). The early genes EF-la. Xbra. Xwnt-B and Mix. 1 are activated in the nuclear transplants. The increased level of endogenous EF.la tran. scripts in embryos compared to unfertilized eggs indicates the activation of zygotic transcription. The expression of EF-1a. Xbra, gsc. Xwnt-B and Mix. 1 shows that zygotic transcription of early genes has been activated in transplanted cultured cell nuclei. Thus. the transplanted nuclei have been reprogrammed to adopt to a new pattern of transcription.
	Fig. 7. In situ hybridization of nuclear transplant embryos using an Xbra probe. In situ hybridization was performed on sections of control embryos (AI and gastrula stage nuclear transplant embryos derived from eel/line 2.14 (8), using a digoxygenin-Iabeled anti~sense Xbra probe. Note the positive staining in region of rhe in vagina ring marginal zone. The dorsal side is to the right.
	Fig. 8. Immunostaining of sections of equatorial explants derived from nuclear transplants. The equatorial regions of an early gastrula control embryo (AI or of a nuclear transplant embryo derived from cell Ime 2. 74 fBI were explanted and cultured until siblmg embryos from fertilized eggs has reached stage 26. The explants were sectioned and stained with the muscle-specific antibody 12/101 (dark blue), and with the notochord-specdlc antibody MZ15 (red), The section of the nuclear transplant embryo is positive for muscle and notochord as is the case of the control, Indicating that mesodermal differentiation has occurred in the correct germ layer of nuclear transplant embryos.