Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

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。     

Chironomid midges: a forgotten model of developmental biology research

For more than a century, embryologists have been exploring various model systems to gain insights into developmental processes. This article presents an overview of the role of chironomid midges in embryology research since their introduction as model organisms in the 19th century. We present the vestiges of bibliography since the days of Weismann (1834–1914), who raised preliminary queries to unravel many unique features of insect embryogenesis using midges as a crucible. Unfortunately, over the years, chironomid midges got lost into obscurity as a model for developmental biology, which is evident from the paucity of developmental biology–related literature on midges in the past decades. Through this essay, the authors intend to share reminiscences of the heydays of chironomid research with the wider community of zoologists with an aim of reviving chironomid embryology. Midges not only possess the basic qualities essential for an ideal model system, but being one of the ancestral dipteran stocks, they can also prove an excellent test system for evo‐devo, transgenetic, and embryogenomic investigations that utilize methodologies at the interface of developmental biology and high‐throughput molecular genetic and genomics approach. An introspection of re‐introducing chironomid midgesas model system will be rewarding for the contemporary developmental biologists.     

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Background

The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results

Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions

The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.     

piggyBac转座系统在哺乳动物及其细胞中的研究进展

piggyBac（PB）转座系统来源于昆虫鳞翅目,属于真核生物的第二类转座系统,主要采取＂剪切粘帖＂机制发生转座。PB系统转座效率高,宿主范围广,广泛应用于昆虫等低等生物的基因转移及突变筛选。近年来,研究发现PB系统在哺乳动物及其细胞中也具有高效的转座活性,已在动物基因组功能研究、基因转移及诱导多能干细胞等领域得到了广泛应用。本文就PB系统近年来在哺乳动物及其细胞中的研究进展、应用前景及存在问题进行了综述。     

Tc1/Mariner转座子超家族的研究进展

随着高通量测序技术的迅猛发展,越来越多的生物基因组注释结果表明：转座子几乎存在于所有生物的基因组中,是大多数生物基因组的重要组分。其中,Tc1/Mariner转座子是自然界中分布最广泛的一类DNA转座子超家族,在自然界已经发现14个有活性的Tc1/Mariner转座子(如Minos,Mos1等),另外通过分子重构也获得高活性的人工转座子,如睡美人转座子(Sleeping Beauty, SB)。SB和Mos1等转座子作为基因转移载体已被广泛应用于转基因、基因捕获和基因治疗等领域的研究中,并取得了很好的应用效果。本文将重点综述Tc1/Mariner转座子的结构、分类、分布、转座机制、活性转座子的挖掘,及其在转基因、基因捕获和基因治疗等研究领域的应用。     

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。     

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     

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.