家蚕转基因载体pBacA3EG的构建及其表达

以家蚕Bombyx mori肌动蛋白A3(actin 3)启动子、增强性绿色荧光蛋白(enhanced green fluorescent protein, EGFP)基因及SV40的多聚腺苷酸识别序列为元件,经多次克隆,将其插入到piggyBac转座载体中。经PCR、酶切鉴定及测序表明各元件已按正确的方式插入到piggyBac载体中。将构建好的piggyBac表达载体显微注射到胚盘形成前期的蚕卵中,在胚胎早期发育的第3天,通过体视荧光显微镜检测到蚕卵内发出较强的绿色荧光。结果表明该载体构建正确且能在蚕卵中进行表达。家蚕转基因载体的体外瞬时表达不但是成功进行家蚕转基因所必需的第一步,而且其自身也可以应用于基因的功能研究,为家蚕后基因组研究奠定了基础。     

A novel hairless mouse model on an atopic dermatitis-prone genetic background generated by receptor-mediated transgenesis

Current mouse models for atopic dermatitis (AD) have a serious drawback, being the existence of dense hair on the body. Thus, a hairless animal model on an AD-prone genetic background will be a powerful tool to investigate the basis of and therapy for this complex disease. We applied the Toxin Receptor-mediated Cell Knockout (TRECK) method to generate a hairless transgenic (Tg) mice on the NC/Nga background, an AD-prone inbred strain. A minigene with the mouse Keratin71 (Krt71) promoter and human diphtheria toxin receptor, which intrinsically functions as the heparin-binding EGF-like growth factor, was introduced into the pronucleus of NC/Nga oocytes. Unexpectedly NCN24, one NC/Nga Tg line, showed a dominant hairless phenotype without diphtheria toxin administration. Furthermore, the atopic dermatitis-like predisposition and IgE elevation was observed in both NCN24 and the NC/Nga wildtype strain. NCN24 mice, which we have newly developed, will be useful to assess drugs for AD therapy, being able to monitor skin inflammation without shaving. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A+C+0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A+C+0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Transgenic analysis of the physiological functions of Mahogunin ring finger‐1 isoforms

Mahogunin Ring Finger‐1 (Mgrn1) null mutant mice have a pleiotropic phenotype that includes the absence of yellow hair pigment, abnormal head shape, reduced viability, and adult‐onset spongiform neurodegeneration. Mgrn1 encodes a highly conserved E3 ubiquitin ligase with four different isoforms which are differentially expressed and predicted to localize to different subcellular compartments. To test whether loss of specific isoforms causes different aspects of the mutant phenotype, we generated transgenes for each isoform and bred them onto the null mutant background. Mice expressing only isoform I or III appeared completely normal. Isoform II rescued or partially rescued the mutant phenotypes, whereas isoform IV had little or no effect. Our data show that different Mgrn1 isoforms are not functionally equivalent in vivo and that the presence of only isoform I or III is sufficient for normal development, pigmentation, and neuronal integrity. genesis 47:524–534, 2009. © 2009 Wiley‐Liss, Inc.     

Developmental regulation of the composite CAG promoter activity in the murine T lymphocyte cell lineage

Promoter selection is of utmost importance for the study of in vivo gene function using transgenic models. In the present study, we have analyzed the expression of the GFP marker under the control of the composite CAG promoter in the lymphoid compartment of several transgenic mouse strains. Despite the ability of the CAG promoter to drive gene expression in almost all tissues examined to date, its activity appears to be developmentally regulated within the T lymphocyte cell lineage. In particular, CD4 and CD8‐expressing, thymic immature T cells displayed lower levels of the GFP marker when compared with both bone marrow precursors and mature circulating T cells, suggesting a transient downregulation of CAG activity during T cell development. Alternative promoters may therefore be preferred for the study of T cell development in vivo using a transgenic approach. genesis 47:799–804, 2009. © 2009 Wiley‐Liss, Inc.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

家蚕卵显微注射操作对蚕卵孵化及畸形蚕发生的影响

早期胚胎显微注射是目前获得转基因家蚕Bombyx mori的主要途径。显微注射操作对蚕卵的损伤导致注射后的蚕卵孵化率降低, 是家蚕转基因工作的主要障碍之一。本研究对不同卵龄的蚕卵进行了开孔或注射实验, 并对产后5 h的蚕卵上背侧、 腹侧、 前极、 后极和中央等5个不同的位置进行了开孔实验, 调查了卵孵化率和体形异常蚕的产生情况。结果表明： 较早卵龄期的注射或从蚕卵背侧的注射可以获得高的孵化率。腹侧注射产生大量的体形异常蚕而背侧注射的蚕完全正常。通过调整注射时期和注射位置避开上述影响可以减少死卵和畸形蚕, 提高孵化率。本研究为改进家蚕转基因操作技术提供了有效的参考。     

Inherited haemolytic anaemia created by insertional inactivation of the α-spectrin gene

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.     

Transgene excision in zebrafish using the phiC31 integrase

Genome engineering strategies employing site‐specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase‐estrogen receptor hormone‐binding domain variant fusion protein catalyzes attB‐attP recombination in zebrafish embryos in a 4‐hydroxytamoxifen‐dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish. genesis 48:137–143, 2010. © 2010 Wiley‐Liss, Inc.