转基因斑马鱼分析胰岛β-细胞发育情况(英文)
ANALYSES OF β-CELL DEVELOPMENT IN TRANSGENIC ZEBRAFISHW ITH A CONSTRUCT OF INSULIN PROMOTER AND GREEN FLUORESCENT PROTEIN
-
摘要: 斑马鱼的个体小、高产和体外受精等特点使其已经迅速成为研究脊椎动物器官发育和人类疾病的模式生物之一。我们建立了一个转基因斑马鱼动物模型来研究胰岛β-细胞的发育。首先，构建了斑马鱼胰岛素(Insulin，INS)启动子与绿色荧光蛋白(GFP)组成的表达载体，命名为INS:GFP。其次，将质粒在斑马鱼1-细胞期注射到细胞质内。最后我们成功获得了生殖系稳定遗传胰岛素转基因斑马鱼，在成鱼和幼鱼期均可以通过GFP标记β-细胞。通过方便的荧光筛选，我们观察到胰岛在受精后18h开始形成，1-5d后由初始的脊索中线两侧向右迁移。从我们构建的胰岛素转基因斑马鱼，可以直观判断胰岛的发育情况，为研究胰岛的发育、损伤和再生提供了一个简便和直观的新型工具。Abstract: W ith many advantageous features such as small size, high production and fertilization in vitro, zebrafish has become a kind of themodel creature for the investigation of vertebrate development and human diseases. Sowe set up a transgenic zebrafish model to investigate the β-cell development1 Firstly, we obtained an INS: GFP construction that contains the zebrafish insulin (INS) promoter and green fluorescent protein (GFP).Secondly, we injected the construction into the cytoplasm of one-cell-stage embryos. F inally, we gained germline INS transgenic zebrafish that d isplayed highly specific β-cell expression ofGFP in both larvae and adult1 By following GFP expression, pancreas formation was detected at the 18h post-fertilization in the earliest time points between the second bilateral som ites, a group of GFP-positive cells located from ventral to the notochord. From day 1 to 5 post-fertilization,the number of insu lin/GFP expressing cells migrated and formed a right organ. Thus, ourworks demonstrated that the transgenic line provided a conven ient and d irect expermi ental tool in analyzing endocrine pancreas development, injury and recovery.
-
Keywords:
- Zebrafish /
- β-cell /
- Green F luorescent Protein (GFP) /
- Insulin
-
-
[1] Shapiro A M, L akey JR, Ryan E A, et al. Islet transplantation in seven patientsw ith type. diabetesmellitus using a glucocort-icoid-free immunosuppressive regimen [J]. N EnglJ M ed, 2000, 343: 230-238
[2] DorY, Brown J, M artinezO I, et al. Adult pancreatic beta-cells are formed by sel-f duplication rather than stem-cell differentiation[J]. N ature, 2004, 429: 41-46
[3] Porat S, DorY. N ew sources of pancreatic beta cells [J]. Curr D iab Rep, 2007, 7: 304-308
[4] Huang H, VogelS S, Liu N, et al. Analysis ofpancreatic development in living transgenic zebrafish embryos [J]. Mol Cell Endocrinol, 2001, 177: 117-124
[5] Rembo ldM, LahiriK, FoulkesN S, et al. Transgenesis in fish: efficient selection of transgenic fish by co-injection w ith a fluorescent reporter construct [J]. N at Protoc, 2006, 1: 1133-1139
[6] K inoshitaM, Kani S, O zato K, et al. Activity of themedaka translation elongation factor 1alpha-A promoter examined using the GFP gene as a reporter [J]. Dev Growth D iffer, 2000, 42: 469-478
[7] W esterfield M1 The Zebrafish Book [M]. Institute of N euroscienceU niversity of Oregon. 1995, 16-21
[8] W anH, Korzh S, Li Z, et al. Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter [J]. Exp CellRes, 2006 312: 1526-1539
[9] Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas [J]. Physiol Res, 2001, 50:537-546
[10] Biemar F, Argenton F, Schm idtke R, et al. Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet [J]. Dev Biol, 2001, 230: 189-203
[11] Yee N S, Yusuff S, PackM. Zebrafish pdx. morphant displays defects in pancreas development and digestive organ chirality, and potentially identifies a multipotent pancreas progenitor cell [J]. Genesis, 2001, 30: 137-140
[12] K im H J, Sumanas S, Palencia-Desai S, et al. G enetic analysis of early endocrine pancreas formation in zebrafish [J]. Mol Endocrinol,2006, 20: 194-203
[13] W ardA B, W arga R M, P rince V E. O rigin of the zebrafish endocrine and exocrine pancreas [J]. D ev Dyn,2007, 236: 1558-1569
[14] SpederP, Petzo ldt A, Suzanne M, et al. Strateg ies to establish left/right asymmetry in vertebrates and invertebrates [J]. Curr Opin GenetD ev, 2007, 17: 351-358
[15] Aylsworth A S. C linical aspects of defects in the determ ination of laterality [J]. Am J M ed Genet, 2001, 101:345-355
[16] P isharath H. Validation of nitroreductase, a prodrug-activating enzyme, mediated cell death in embryonic zebrafish (Danio rerio) [J]. Comp M ed, 2007, 57: 241-246
[17] P isharath H, Rhee JM, SwansonM A, et al. T argeted ablation of beta cells in the embryonic zebrafish pancreas using E. colin-itroreductase [J]. M ech Dev, 2007, 124: 218-229
[18] Curado S, Anderson R M, Jungblut B, et al. Conditional targeted cell ablation in zebrafish: a new too l for regeneration studies [J]. D ev Dyn, 2007, 236: 1025-1035
[19] M cGonnell IM, Fowkes R C. F ishing for gene function-endocrine modelling in the zebrafish [J]. J Endocrinol, 2006, 189:425-439
计量
- 文章访问数: 884
- HTML全文浏览量: 5
- PDF下载量: 432
下载: