Human sickle hemoglobin in transgenic mice

DNA molecules that contain the human alpha- and beta s-globin genes inserted downstream of erythroid-specific, deoxyribonuclease I super-hypersensitive sites were coinjected into fertilized mouse eggs and a transgenic mouse line was established that synthesizes human sickle hemoglobin (Hb S). These animals were bred to beta-thalassemic mice to reduce endogenous mouse globin levels. When erythrocytes from these mice were deoxygenated, greater than 90 percent of the cells displayed the same characteristic sickled shapes as erythrocytes from humans with sickle cell disease. Compared to controls the mice have decreased hematocrits, elevated reticulocyte counts, lower hemoglobin concentrations, and splenomegaly, which are all indications of the anemia associated with human sickle cell disease.     

Globin composition and synthesis of hemoglobins in developing fetal mice erythroid cells

Fetal mouse erythropoiesis proceeds initially in yolk-sac blood islands (8 to 12 days) and, subsequently, in liver (12 to at least 16 days). Yolksac cells synthesize three hemoglobins, Hb E(I), Hb E(II) and Hb E(III). Hb E(I) has x- and y-globin chains; Hb E(II) has alpha and y; HB E(III), alpha and z. No detectable beta-globin is formed in these cells. Liver erythroid cells form only adult hemoglobin, composed of alpha- and beta-chains.     

Hemoglobin Lepore trait: globin synthesis in bone marrow and peripheral blood

There was decreased synthesis of the beta-globin chain in the peripheral blood, and equal synthesis of alpha- and non-alpha-chains in the bone marrow of three patients with hemoglobin Lepore trait, similar to the findings in patients with heterozygous beta-thalassemia. There is a relative instability of the synthetic mechanism for normal beta-chain in these patients.     

Correction of murine beta-thalassemia by gene transfer into the germ line

A murine beta-thalassemia was corrected by the transfer of cloned beta-globin genes into the mouse germ line. The cloned mouse beta maj-globin gene or the cloned human beta-globin gene was introduced into mice deficient in beta-globin synthesis because of a deletion of the beta maj-globin gene. Both introduced genes produced functional beta-globin chains, leading to a reduction in one case, and elimination in another case, of the anemia and associated abnormalities of the red blood cells.     

Hemoglobin Gun Hill: deletion of five amino acid residues and impaired heme-globin binding

Hemoglobin Gun Hill, a new variant of adult hemoglobin, was found in a Caucasian and one of his three daughters. The abnormal hemoglobin had only half of the expected number of heme groups. Five amino acid residues appeared to be missing from the beta-globin chains. These residues occur in linear sequence in normal beta-chains in a region involved in heme-globin binding. A deletion of five amino acids in the beta-chains of hemoglobin Gun Hill is postulated. The most likely mechanism for the origin of such a hemoglobin variant would appear to be unequal crossing-over during meiosis.     

Mini-mouse: disruption of the pygmy locus in a transgenic insertional mutant

A founder transgenic mouse harbored two different integration patterns of a transgene at the same locus, each of which gave rise to a similar autosomal recessive mutation. Mice of the mutant phenotype were of small stature but had normal levels of growth hormone. The disrupted locus was cloned, and a genetic and molecular analysis showed that the insertional mutants were allelic to a spontaneous mutant, pygmy. The mice should be a useful model for the growth hormone-resistant human dwarf syndromes and could lead to a greater understanding of the pathways involved in growth and development.     

诱导性免疫共刺激分子转基因小鼠模型的建立

利用RT—PCR方法从人扁桃体的激活T细胞mRNA中扩增出诱导性免疫共刺激分子(ICOS)cDNA,继而将ICOS基因插入到pEGFP—N3中得到含ICOS和GFP融合蛋白的pEGFP—ICOS。将外源基因pEGF、P—ICOS注射至FVB小鼠原核中,其胚胎移植到同期发情的假孕受体产出后代,经PCR和Southern检测获得阳性转基因小鼠。试验共注射移植胚胎117枚,出生小鼠43只,检出阳性小鼠2只,该转基因已稳定遗传至F3代,说明建立了ICOS转基因小鼠模型。     

Hemoglobin types of Macaca irus and Macaca mulatta monkeys

Hemoglobin of 30 Macaca mulatta monkeys and of 15 Macaca irus monkeys consisted of one electrophoretic component similar to human hemoglobin A. Twenty-one M. irus monkeys had two types of hemoglobin. In 20 animals the hemoglobin resembled human hemoglobin AJ, and in one animal it resembled human hemoglobin AI.     

Autonomous developmental control of human embryonic globin gene switching in transgenic mice

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.     

Construction of a universal recombinant expression vector that regulates the expression of human lysozyme in milk

The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here, we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome (BAC) vectors. The BAC of mouse whey acidic protein gene (mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene (hLZ) was then inserted into the vector by a digestion-ligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb hLZ genomic DNA, and 8.0 kb mWAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme (rhLZ) in milk. The highest expression level of rhLZ was 0.45 g·L⁻¹, and rhLZ exhibited the same antibacterial activity as native hLZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.     

转基因番茄植株Mi1.2表达水平的Real Time PCR检测体系的建立

[目的]为转基因番茄植株的高通量筛选奠定基础。[方法]利用CTAB法提取番茄叶片总RNA进行Real Time PCR扩增,分析转Mi1.2基因的番茄植株表达水平的检测体系。[结果]提取RNA的A260/A280为1.78~1.88,RNA无明显降解。在严谨扩增条件下,引物SYBR2的扩增效率高于SYBR1。Mg2+的适宜浓度为2.0 mg/L。Real Time PCR扩增产物具有良好的特异性,熔解曲线特异峰出现在84.5℃附近,在熔解曲线略低于83℃附近有极微弱的非特异峰。因此在定量反应中信号检测步骤应放在84℃。以4种不同模板分子数条件下扩增曲线Ct值得到的回归方程为Y=-3.78×log(copynumber)+39.50,相关系数为0.998。[结论]该试验获得的Real Time PCR体系可用于转基因植株表达水平的检测。     

转基因水稻深加工产品两种荧光定量方法的比较研究

根据转基因水稻中外源基因Cry1A(B)和内源基因SPS设计SYBR Green I引物、TaqMan引物和荧光探针,以内源基因SPS作为内参照,使用两种FQ-PCR方法对水稻深加工产品中转基因水稻的含量进行定量检测.建立了转基因水稻标准品Cry1A(B)和SPS之间的△Ct与样品中转基因水稻含量百分比之间的校正曲线和...     

Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin mu

Antibody-producing cells display a special form of regulation whereby each cell produces immunoglobulin from only one of its two sets of antibody genes. This phenomenon, called allelic exclusion, is thought to be mediated by the product of one heavy chain allele restricting the expression of the other. Heavy chains are synthesized in two molecular forms, secreted and membrane bound. In order to determine whether it is specifically the membrane-bound form of the immunoglobulin M (IgM) heavy chain (mu) that mediates this regulation, transgenic mice were created that carry a human mu chain gene altered so that it can only direct the synthesis of the membrane-bound protein. The membrane-bound form of the human mu chain was made by most of the B cells in these animals as measured by assays of messenger RNA and surface immunoglobulins. Further, the many B cells that express the human gene do not express endogenous mouse IgM, and the few B cells that express endogenous mouse mu do not express the transgene. Thus, the membrane-bound form of the mu chain is sufficient to mediate allelic exclusion. In addition, the molecular structures recognized for this purpose are conserved between human and mouse systems.     

Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice

Two transgenic mice were obtained that contain in their chromosomes the complete hepatitis B virus (HBV) genome except for the core gene. These mice secrete particles of HBV surface antigen (HBsAg) in the serum. In one mouse, HBV DNA sequences that had integrated at two different sites were shown to segregate independently in the first filial generation (F1) and only one of the sequences allowed expression of the surface antigen. Among these animals the males produced five to ten times more HBsAg than the females. A 2.1-kilobase messenger RNA species comigrating with the major surface gene messenger RNA is expressed specifically in the liver in the two original mice. The results suggest that the HBV sequences introduced into the mice are able to confer a tissue-specific expression to the S gene. In addition, the HBV transgenic mice represent a new model for the chronic carrier state of hepatitis B virus infection.     

Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice

Primary and secondary hypertriglyceridemia is common in the general population, but the biochemical basis for this disease is largely unknown. With the use of transgenic technology, two lines of mice were created that express the human apolipoprotein CIII gene. One of these mouse lines with 100 copies of the gene was found to express large amounts of the protein and to be severely hypertriglyceridemic. The other mouse line with one to two copies of the gene expressed low amounts of the protein, but nevertheless manifested mild hypertriglyceridemia. Thus, overexpression of apolipoprotein CIII can be a primary cause of hypertriglyceridemia in vivo and may provide one possible etiology for this common disorder in humans.     

BMP4对小鼠毛囊数量的影响

利用PolⅡ型人角蛋白14基因的启动子K14驱动eGFP-shRNA整合转录本的表达方法,制备在皮肤组织中高效表达靶向BMP4基因的siRNA转基因小鼠模型。利用Northern杂交的方法验证siRNA在转基因小鼠皮肤中高表达,但未从mRNA水平分析BMP4基因的表达变化。以建立的转基因小鼠模型为基础,检测小鼠皮肤组织中BMP4基因的表达变化,并观察妊娠18.5d(E18.5)转基因胎鼠与同窝阴性胎鼠背部皮肤组织切片中毛囊数量的变化,选择5个不同的视野进行毛囊数量计数后统计分析。结果表明,转基因小鼠皮肤组织中BMP4基因的表达下调,同时毛囊数量显著增加。表明利用融合表达载体制备转基因小鼠实现目的基因RNAi的方法可以有效地抑制动物体内目的基因的表达,同时也从胎鼠毛囊数量分析统计的结果发现,该基因对小鼠毛囊的发生发育有一定的影响。     

Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

In this study, we introduced the bovine immunoglobulin μ heavy-chain gene (the orphaned gene on BTA11) into mouse germline cells. Bovine IgM was highly expressed in selected transgenic lines, and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain (IgH) genes in these lines. The forced expression of bovine IgM resulted in reduced numbers of pro- and pre-B cells but increased the number of immature B cells in the transgenic mice. Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen, but most of the cells are arrested at the T1 transitional B cell stage, leading to a significantly lower number of T2 transitional and mature B cells in the spleen. Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased, the IgA levels were slightly increased compared to the WT mice. The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM, suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells. These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3 (CDR3) sequences in their VH repertoire and Vκ repertoire but exhibited an increased frequency of unique CDR3 in their Vλ repertoire. Compared to the WT mice, the transgenic mice had a significantly higher percentage of mouse IgM-expressing B cells that expressed λ chains. Finally, we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.     

Expression of a microinjected porcine class I major histocompatibility complex gene in transgenic mice

A porcine class I major histocompatibility complex (SLA) gene has been introduced into the genome of a C57BL/10 mouse. This transgenic mouse expressed SLA antigen on its cell surfaces and transmitted the gene to offspring, in which the gene is also expressed. Skin grafts of such transgenic mice were rejected by normal C57BL/10 mice, suggesting that the foreign SLA antigen expressed in the transgenic mice is recognized as a functional transplantation antigen.     

Human-specific gain of function in a developmental enhancer

Changes in gene regulation are thought to have contributed to the evolution of human development. However, in vivo evidence for uniquely human developmental regulatory function has remained elusive. In transgenic mice, a conserved noncoding sequence (HACNS1) that evolved extremely rapidly in humans acted as an enhancer of gene expression that has gained a strong limb expression domain relative to the orthologous elements from chimpanzee and rhesus macaque. This gain of function was consistent across two developmental stages in the mouse and included the presumptive anterior wrist and proximal thumb. In vivo analyses with synthetic enhancers, in which human-specific substitutions were introduced into the chimpanzee enhancer sequence or reverted in the human enhancer to the ancestral state, indicated that 13 substitutions clustered in an 81-base pair module otherwise highly constrained among terrestrial vertebrates were sufficient to confer the human-specific limb expression domain.