青鱼β-actin基因克隆及其启动子功能的初步检测

高保真PCR克隆青鱼β-actin基因开放阅读框和5’端侧翼序列,DNA测序结果表明：青鱼β-actin基因开放阅读框编码一段含375个氨基酸的蛋白,与其他物种actin家族相比较具有高度保守性。青鱼β-actin与鲤鱼、草鱼及斑马鱼的同源性均为100％,而与人和Norway鼠β-actin的同源性均为99.2％,与鸡和Kenyan爪蟾β-actin的同源性分别为98．9％和98．1％。将青鱼β-actin基因5’端启动调控区插入不含启动子的pEGFP1载体构建青鱼β-actin启动子／EGFP表达载体,与第一次卵裂之前显微注射该重组质粒入泥鳅受精卵,同时也用该重组质粒转染HeLa细胞系。观察结果表明：GFP在50％的泥鳅胚胎和2／3的HeLa细胞有所表达。GFP在泥鳅胚胎的各个部分均有表达,且在某些胚胎中GFP的表达遍布全身。因此,以EGFP为报告基因证实了青鱼β-actin基因启动子为一种非特异性表达的启动子。     

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos

The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin. The experiments reported here were designed to determine if the inclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identification using a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryos were microinjected with GFP reporter gene under the control of three different promoters and flanked or not by three different MAR elements. Transgene expression profiles were followed by direct visualization of GFP in cultured microinjected embryos. Embryos at different developmental stages were classified according to their GFP expression and groups with the same expression pattern were transferred into oviducts of pseudopregnant female mice. Fetuses were collected between days 12–15, and their genomic DNA was purified and analyzed to detect transgene integration. We did not find any statistically significant difference between the percentage of transgenic fetuses produced from GFP-positive or GFP-negative embryos transferred at 4-cell, morula, or blastocyst stage. However, when MAR elements were included in the construct, we found that GFP-positive embryos transferred at the 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, but MAR sequences did not completely eliminate false positives.     

Cloning of Black Carp β-actin Gene and Primarily Detecting the Function of Its Promoter Region

A 3 338 bp DNA fragment including the open reading frame and 5′-flanking region of β-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP₁ vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.     

异源Oct-4基因启动子在猪、兔和小鼠早期胚胎中的时空表达活性

Oct-4是一种哺乳动物早期胚胎中特异表达的转录因子,它与细胞多能性的维持有关．异源Oct-4基因在早期胚胎中的表达模式尚不明确．构建了一个以完整的牛Oct-4调控区指导GFP表达的转基因结构pOct-4(p)-GFP,通过单精子注射的方法将其导入猪、兔和小鼠的受精卵中,分析其在胚胎发育过程中的表达情况．结果显示：牛Oct-4启动子驱动的GFP基因在3个物种的2-细胞胚胎就已经开始表达,在囊胚期表达加强且只特异表达于内细胞团中,而不表达于滋养层．研究表明：牛的Oct-4启动子在其他物种中也具有表达活性,异源性Oct-4启动子在不同物种的早期胚胎中具有相似的表达模式．     

Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.     

Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo. Received December 21, 1998; accepted March 31, 1999.     

Direct DNA delivery into zebrafish embryos employing tissue culture techniques

Summary: The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24–48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources. genesis 31:1–5, 2001. © 2001 Wiley‐Liss, Inc.     

Drosophila alcohol dehydrogenase: a novel reporter gene for use in mammalian embryos.

A reporter gene construct containing the Drosophila alcohol dehydrogenase (ADH) gene under regulation of the Rous sarcoma virus long terminal repeat (RSV LTR) was microinjected into mouse zygote pronuclei. ADH activity after injection of the RSV-ADH construct was visualized in cultured embryos by means of a simple histochemical enzyme assay. The RSV LTR was an efficient promoter that led to abundant ADH activity at all stages of preimplantation development. There was a high incidence of mosaicism among stained embryos.     

Germline transformation of the sawfly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector

A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous helper plasmid containing an active piggyBac transposase gene into the posterior end of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera: Symphyta). These injected eggs, which developed as haploid male embryos upon artificial activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid adult males. These G(0) haploid males were individually mated to diploid females. The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1) diploid females were activated artificially, and the resultant embryos were examined for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2) haploid embryos segregated to GFP-positive and -negative individuals. By mating the G(2) adult haploid males individually to diploid females, stocks were established in which the piggyBac construct was stably integrated into the genome, as evidenced by GFP expression and Southern blot hybridization. The piggyBac transposition occurred at its canonical target TTAA sequence. These results, which demonstrate the first successful stable transposon-mediated germline transformation in Hymenoptera, will expand the usefulness of the piggyBac vector.