摘    要

  随着人类基因组计划（HGP）的顺利完成，后基因时代的生物学研究迫切需要有效的基因功能分析方法。基因敲除模型的应用，为研究基因的功能和寻找新的治疗人类疾病的干预措施提供了有力支持。

  Lhx4基因是LIM同源盒基因家族中的一员，其蛋白产物是一种神经内分泌转录因子，是垂体发育和运动神经元特化所需要的。基因敲除实验表明Lhx4-/-小鼠垂体前叶六种细胞系中有四种缺失，它们是促性腺细胞、催乳细胞、生长激素细胞、促甲状腺素细胞、而且Lhx4的功能不能由Lhx4补偿。表明Lhx4是垂体形成和垂体前叶激素分泌细胞类型特化所必需的。红色荧光蛋白(drFP583 )是人们从珊瑚虫Discosoma gen中克隆的一种与绿色荧光蛋白(GFP)同源的荧光蛋白，在紫外光的照射下可发射红色荧光，有着广泛的应用前景;但它自身的缺点如寡聚化、成熟缓慢等限制了它的进一步应用。因此，人们对它进行了一系列修饰和改进，得到了寡聚化程度低(甚至单体)和成熟速率快的突变体，Clontech公司已将一种突变体商业化，命名为DsRed。与GFP相比，DsRed的激发和发射波长较长，其发射峰位于培养基、组织培养器材及细胞成分等产生的荧光背景范围之外，具有较高的信噪比;而且在细胞内荧光转换效率高，更易检测。红色荧光蛋白(DsRed) 的突变体有mBanana、mOrange、tdTomato、mTangerine、mStrawberry 和mCherry。在所有光谱型中，亮度最强的荧光蛋白是串联形式的二聚体Tomato（dimeric Tomato, dTomato）。它是一种原始的水果蛋白的橙色衍生物。dTomato来源于一种被命名为dimer2的中间体，这种中间体是在四聚体DsRed蛋白崩解过程中产生的。这种变体包含GFP的N末端和C末端的前后各七个氨基酸残基，这样可以提高与融合蛋白的兼容性并降低潜在的定位假象。串联二聚体包含dTomato的两个拷贝，由12个氨基酸残基的接头链接。由于有一对发色团，导致tdTomato亮度极强并且有得天独厚的光稳定性。85195

  本文运用PCR技术，根据小鼠基因的序列设计用于基因型鉴定的引物，取材，提取DNA，PCR目的片段，分析PCR结果，相应的组织器官发育阶段联系起来，指导转基因小鼠作为工具鼠应用于条件性敲除中，有助于研究垂体发育和运动神经元特化。

毕业论文关键词： PCR扩增 ;  生长发育;  基因型鉴定;  tdTomato

Abstract

    With the successful completion of the human genome project (HGP), the biological research in the post genomic era is in urgent need of effective gene function analysis。 The application of gene knockout model provides strong support for the study of gene function and the search for new interventions for the treatment of human diseases。

    Lhx4 is a member of the LIM gene homologous box gene family, its protein product is a nerve endocrine transcription factor, is pituitary development and motor neuron specialization required。 Experimental results show that Lhx4-/- gene knockout mouse pituitary cell line six with four missing, they are gonadotroph cells, growth hormone, prolactin, TSH cells, and cell function can not be compensated by Lhx4 。 That Lhx4 is formed and pituitary hormone secretion in the anterior pituitary cell types required for specialization。 The red fluorescent protein (drFP583) is cloned from coral Discosoma Gen with a green fluorescent protein (GFP) fluorescent protein homologue, emits red fluorescence in the UV irradiation, has a broad application prospect; but its disadvantages such as oligomerization, mature and slow limits further application of it。 Therefore, a series of modifications and improvements have been carried out, and the mutants with low degree of oligomerization (even monomer) and rapid maturation rate have been obtained。 Clontech has commercialized a mutant named DsRed。 Compared with GFP, the excitation and emission wavelengths longer DsRed, the emission peak located in the range of background fluorescence equipment and components of the cell culture medium, the organization, has a high signal-to-noise ratio; and the intracellular fluorescence conversion efficiency is high, easy to detect。 Red fluorescent protein (DsRed) mutants have mBanana, mOrange, tdTomato, mTangerine, mStrawberry and mCherry。 Among all the spectral types, the most intense fluorescent protein is the two Tomato (dimeric Tomato, dTomato) in the tandem form。 It is an orange derivative of the original fruit protein。 DTomato is derived from an intermediate, named dimer2, which is produced during the disintegration of the four mer DsRed protein。 This variant contains seven amino acid residues at the N terminus and the C end of the GFP, which can enhance the compatibility with the fusion protein and reduce the potential for false localization。 The tandem two contains two copies of the dTomato, which are linked by the attachment of the 12 amino acid residues。 Because has a pair of chromophores, leading to tdTomato brightness strong and light stability, richly endowed by nature。