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Transgenic Cashmere Goat of Hair Follicle Cells Specific Expression Thymosin Beta 4

Author: JinYong
Tutor: LiuDongJun；GuoXuDong
School: Inner Mongolia University
Course: Zoology
Keywords: Transgenic cashmere goat Specific expression of hair follicle cells Thymosin β4 Fetal fibroblasts Somatic cell nuclear transfer PCR.
CLC: S827
Type: Master's thesis
Year: 2011
Downloads: 70
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Abstract

Animal transgenic technology is one of the most rapid development of the 21st century's biological high-tech biological engineering technology in cytogenetics, molecular genetics, embryonic development, and recombinant DNA technology foundation. Since the 1997 cloned sheep \This study used a SCNT transgenic methods, i.e. follicle specific expression promoter pKAP6-1 boot thymosin β4 (thymosin beta 4, Tβ4) gene, neomycin resistance gene and the red fluorescent protein gene as a double selection mark, build Tβ4 expression vector is specifically expressed in the hair follicle cells; transfection technique using in vitro expression vector into the cashmere goat fetal fibroblast cells stably expressing red fluorescent protein cloned cells were screened by G418; somatic cell nuclear transfer technology to prepare velvet goat cloned transgenic embryos; through embryo transfer, production of transgenic cashmere goat. Of this research is transferred to the cultivation of genetically cashmere goats, and to explore the role of Tβ4 in the the cashmere goats hair follicle growth cycle regulation provide a research foundation. Follicle cell-specific expression vector and amplified by PCR and RT-PCR method from the sheep total DNA and total RNA of the cashmere goat keratin associated protein 6-1 gene (KAP6-1) of the promoter region sequence and velvet The goat thymosin p4 (Tβ4) coding region of the cDNA sequence, clone skeleton pMD19T construct an intermediate cloning vector p19TKT. The CMV promoter sequence connection pDsRed2-1 expression in the framework of the red fluorescent protein report gene (Red2) upstream Kpn Ⅰ the Sma Ⅰ site points constitute pCDsRed2; pCDsRed2 by Sac Ⅰ Hind Ⅲ digestion, p19TKT the KT fragments connected constitute a hair follicle cells specific express carrier pCDsR- KT. 2. Cashmere goats fetal fibroblasts in vitro culture and gene transfection by tissue explant attachment method successfully isolated the cashmere goat fetal fibroblast cells using liposomes Lip2000TM will build a good hair follicle-specific expression vector pCDsR-KT into velvet goat fetal fibroblasts genome. Again using the G418 drug and red fluorescent protein expression double screened positive cell clones the positive transgenic cell clones derived cells, fetal sex and integration of exogenous genes in the cell genome, and were identified by PCR method. Growth curve drawn through transgenic / non-transgenic cell lines with chromosome number analysis, assessment of genetically modified cell activity. Into fiber cells after liposomal transfection, screening, and received while having a G418-resistant cells expressing red fluorescent protein clone, exogenous target gene has been stable integration into the cell genome, and transgenic cell lines with the non-transgenic cell lines Cell Growth characteristics of the various periods of the curve remained consistent both cell lines, the normal number of chromosomes (2n = 60) and 76.7% (23/30) and 83.3% (25/30), two types of cells with normal chromosome number was similar . The above analysis shows that the transgenic cells obtained fully used for the next step somatic cell nuclear transfer. Using somatic cell nuclear transfer technology to prepare transgenic cashmere goat embryos slaughterhouse Caprine ovaries, cutting method to collect oocytes and in vitro maturation, mature oocytes by micromanipulation method to remove the nuclear material and the two were fused to form the reconstructed embryos injected transgenic somatic cells, followed by the use of electric shock, reconstructed embryos after in vitro activation in vitro development to the 4-8 cell stage embryo transfer. Cashmere goat in vitro maturation of oocytes was 77.5%, the go the nuclear survival rate was 93.6%, 82.4% fusion rate of reconstructed embryos, in vitro development of cleavage rate was 93.5%, 8 - cells was 24.5%, the blastocyst rate was 14.3%. PCR results showed that exogenous genes stably integrated in the genome of transgenic embryos, fluorescence microscopy stable expression of red fluorescent protein transgenic embryos. Explored the cashmere goat SCNT process efficient in vitro activation method using IA23187 6-DMAP Ionomycin 6-DMAP, 7% ethanol 6-DMAP, IA23187 CHX, the Ionomycin CHX, 7% ethanol CHX treatment in vitro matured oocytes in vitro activate Studies. The study results showed that the the IA23187 6-DMAP and Ⅰ onomycin 6-DMAP treatment oocyte obtain higher blastocyst rate, the two methods are the most suitable method of activation of the cashmere goat oocytes. Transgenic cashmere goat production and identified by PCR using transgenic somatic cell nuclear transfer technology to produce transgenic embryos receptor goat embryo transplant, after puberty observation to determine the number of pregnant ewes and ewe feeding and management during pregnancy, and ultimately 19 Only transgenic cashmere goat. Turn genes cashmere goats after the blood sample extraction and PCR identification, proof of 19 lambs were transferred pCDsR-KT gene cashmere goats, of which 15 Lamb gene group detection to a thymosin β4 gene; while the other four of the Lamb gene group integration have a partial sequence of the carrier pCDsR-KT 3 lambs genome integration of the neomycin resistance gene, the CMV promoter sequences are integrated in the genome of all four of the Lamb.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Goat
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