Regulation of growth hormone-releasing hormone gene expression and biosynthesis

Growth hormone-releasing hormone (GRH) was initially isolated, characterized, sequenced, and cloned from human tumors and subsequently from the hypothalamus of humans and other animal species. Extensive structure-function studies have indicated the amino terminus to be most important for its biologic action, and the primary mechanism of its bioinactivation occurs by cleavage of an amino terminal dipeptide. The GRH gene is expressed primarily in the hypothalamic arcuate nucleus but also in the placenta. Expression of the GRH gene is regulated by growth hormone in a classical feedback manner, with hypophysectomy leading to increased expression that is reversed by growth hormone treatment. GRH gene overexpression in transgenic mice leads to a syndrome similar to that of ectopic GRH secretion with massive pituitary hyperplasia and markedly enhanced growth. The transgenic mouse has been used for studies of GRH biosynthesis and provides a suitable model for the study of precursor processing to the mature hormone.     

Genomic structure, chromosomal localization, and expression pattern of the human LIM-homeobox3 (LHX 3) gene

Lhx3 is a LIM-homeobox protein essential for pituitary development in mice. The human homologue gene spans 7.2 kb and contains 7 exons, including two alternatively spliced first exons. This structure encodes two distinct protein isoforms, LHX3a and LHX3b, that differ exclusively in their amino-terminus. The LHX3 gene was localized at 9q34.2-34.3. The predicted protein sequence is highly homologous to other known Lhx3 proteins, the highest degree of homology being in the conserved domains. The highest expression of LHX3 was found in pituitary gland, spinal cord, and lung. Among different pituitary cell types, corticotrophs appear to express preferentially LHX3b isoform, suggesting a distinct role of the b-form in the development of this cell lineage. Although the human LHX3 gene structure would provide a ground for clarification of the molecular basis of complete anterior pituitary deficiency, we were unable to identify any mutation in the LHX3 gene of 46 such patients.     

Syndromic short stature in patients with a germline mutation in the LIM homeobox LHX4

Studies of genetically engineered flies and mice have revealed the role that orthologs of the human LIM homeobox LHX4 have in the control of motor-neuron-identity assignment and in pituitary development. Remarkably, these mouse strains, which bear a targeted modification of Lhx4 in the heterozygous state, are asymptomatic, whereas homozygous animals die shortly after birth. Nevertheless, we have isolated the human LHX4 gene, as well as the corresponding cDNA sequence, to test whether it could be involved in developmental defects of the human pituitary region. LHX4, which encodes a protein 99% identical to its murine counterpart, consists of six coding exons and spans >45 kb of the q25 region of chromosome 1. We report a family with an LHX4 germline splice-site mutation that results in a disease phenotype characterized by short stature and by pituitary and hindbrain (i.e., cerebellar) defects in combination with abnormalities of the sella turcica of the central skull base. This intronic mutation, which segregates in a dominant and fully penetrant manner over three generations, abolishes normal LHX4 splicing and activates two exonic cryptic splice sites, thereby predicting two different proteins deleted in their homeodomain sequence. These findings, which elucidate the molecular basis of a complex Mendelian disorder, reveal the fundamental pleiotropic role played by a single factor that tightly coordinates brain development and skull shaping during head morphogenesis.     

基于新的CRISPR/Cas9技术实现斑马鱼LHX9基因的快速编辑及对F0突变体性腺发育的初筛

目的:CRISPR/Cas9系统在斑马鱼的反向遗传学中的到了广泛应用,但突变基因的表型观察往往需要在突变鱼系的F1中进行,费时较长。LHX9作为LIM家族的一种转录因子,在胚胎早期的泌尿生殖嵴中有广泛分布;且LHX9基因敲除的小鼠存在性腺发育不良。本研究拟通过一种新的CRISPR/Cas9基因编辑技术,采用四条sgRNA对LHX9基因进行VASA转基因斑马鱼的基因敲除,以观察该基因缺陷对斑马鱼性腺发育的影响。方法:利用新的CRISPR/Cas9技术,设计四条针对斑马鱼LHX9基因3号外显子的20bp的sgRNA,通过非克隆体外转录得到靶位点的四条sgRNA。将以上靶位点的四条sgRNA与Cas9核酸酶蛋白同时注射入单细胞期的斑马鱼胚胎内,利用PCR鉴定突变型类型和突变比例。通过对LHX9基因突变体的VASA转基因斑马鱼进行荧光观察,发现LHX9基因缺陷的斑马鱼性腺发育的情况。结果:靶向Exon 3的四条sgRNA可成功编辑斑马鱼LHX9基因,敲除效率高达82%,Sanger测序发现产生10种不同的移码突变类型。通过该方法对VASA转基因斑马鱼的LHX9基因进行编辑,发现LHX9基因突变导致dph6的的斑马鱼原始生殖细胞增殖和迁移受到影响。结论:基于4条sgRNA注射的CRISPR/Cas9技术,可以快速地产生具有突变表型的G0斑马鱼,具有应用潜力。LHX9基因敲除导致原始生殖细胞的发育和迁移受到影响,提示该基因参与了斑马鱼早期性腺的发育。     

人α-半乳糖苷酶转导克服异种移植HAR的小鼠实验研究

α 半乳糖苷酶可以特异地清除半乳糖α 1,3 半乳糖抗原 (Galα1,3Galantigen) ,此抗原是引起异种器官移植超急性排斥反应 (HyperacuteRejection ,HAR)的主要异种抗原 .将构建好的α半乳糖苷酶转基因载体通过显微注射的方式注入小鼠受精卵 ,培育出了转基因小鼠 .结果表明 ,转基因小鼠的心、肝、肾、脾、肺组织中均有人α 半乳糖苷酶基因的表达 ,其表达可以有效减少小鼠器官表面Galα1,3Gal抗原的表达水平 ,可以降低转基因小鼠脾细胞对补体介导的杀伤作用的敏感性 .研究表明人源α半乳糖苷酶基因可用于研制不表达Galα1,3Gal抗原的转基因动物 ,从而可以降低异种器官移植HAR的反应强度 ,提高移植物的存活期