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.     

Uprooting and Burial of Invasive Alien Plants: A New Tool in Coastal Restoration?

Invasive alien plants are a problem for conservation management, and control of these species can be combined with habitat restoration. Subsoil burial of uprooted plants is a new method of mechanical control, which might be suitable in disturbed habitats. The method was tested in Rosa rugosa (Japanese Rose), an invasive shrub in north‐western Europe with negative effects on coastal biodiversity. Two months after uprooting and burial in dunes of north‐eastern Denmark, 89% of the 58 shrubs resprouted from roots and rhizomes; on average 41 resprouts per shrub. Resprout density was twice as high at former shrub margins compared with the center; resprouts were taller and originated from more superficial soil layers at the margin than in the center. Resprouting was negatively correlated with fragment depth, and no resprouts were observed from greater than 15 cm depth. The number of resprouts increased with fragment dry mass (0.5–168.5 g). After 18 months with harrowing the species was still resprouting, flowering, and fruiting, albeit with no difference between shrub margin and center. Resprouts were taller (26 cm) and coverage was higher (0–4%) after two compared with three times harrowing, whereas no difference was found in cover of native dune species (1–5%). The results show that even small fragments of R. rugosa resprout, and that resprouting persists despite repeated harrowing. Thus, careful subsoil burial of all fragments is necessary, special attention should be paid to the shrub margin, and follow‐up treatments are needed. The effectiveness of the burial method is discussed for restoration of coastal dunes.     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

Unique genomic configuration revealed by microsatellite DNA in polyploid dogroses, Rosa sect. Caninae

An allopolyploid complex with high genomic integrity has been studied. Dogroses transmit only seven chromosomes (from seven bivalents) through the pollen, whereas 21, 28 or 35 chromosomes (from seven bivalents and 14, 21 or 28 univalents) come from the egg cells. Seedlings derived from two interspecific crosses were analysed with flow cytometry and molecular markers to determine ploidy level, mode of reproduction and genomic constitution. Evidence was obtained for the formation of unreduced male and female gametes, which can take part in fertilization (producing seedlings with higher ploidy than the parental plants) or in apomictic reproduction. Random amplified polymorphic DNA (RAPD) and microsatellite analyses indicated that three seedlings (5%) were derived through apomixis, whereas the other 49 were hybrids. Bivalent formation appears to involve chromosomes that consistently share the same microsatellite alleles. Allele-sharing between the maternally transmitted and highly conserved univalent-forming chromosomes reflected the taxonomic distance between different genotypes. The frequently recombining bivalent-forming chromosomes were taxonomically less informative.     

提高狗蔷薇离体培养植株再生频率*

以狗蔷薇( Rosa canina Inermis) 为材料, 以MS 为基本培养基, 通过对不同植物生长调节剂的组合,
大幅度提高了狗蔷薇离体培养植株再生频率。结果表明, 210 mgPL 6-BA+ 01 3 mgPL 2, 4-D 的组合较为适宜,
其不定芽再生率为87%, 增殖率为310; 而CPPU 和2, 4-D 的适宜组合为115 mgPL+ 013 mgPL, 其不定芽再
生率高达93%, 增殖率为510。同时, 研究结果显示, 以MS+ 40 gPL 蔗糖+ 610 gPL 琼脂粉+ 31 5 mgPL
AgNO3 + 115 mgPL CPPU+ 01 1 mgPL 2, 4-D+ 0105 mgPL GA3 作增殖培养基效果最好, 不定芽诱导率为89%,
增殖率为51 5; 利于生根的培养基为1P4MS+ 20 gPL 蔗糖+ 315 gPL 琼脂+ 013%活性碳+ 011 mgPL IBA+ 011
mgPL NAA, 生根率为91%。     

平阴玫瑰花芽分化期叶片内源激素的变化

对平阴玫瑰花芽分化期叶片甲醇提取物进行IAA、ZR、GA₃、ABA的分离、纯化和测定。结果发现,所测的几种激素均表现出明显的变化规律,其中IAA和GA₃在花芽分化期含量逐渐下降,且在分化临界期出现一低峰,而ZR和ABA则完全相反。同时经比较分析得出ABA/GA₃, ABA/IAA,ZR/GA₃,ZR/IAA也表现明显的变化规律,即比值总体趋势是逐渐提高,且均在分化临界期含量出现一飞跃,显然ABA/GA3,ABA/IAA,ZR/GA₃,ZR/IAA在平阴玫瑰的花芽分化过程中起着重要的调控作用,由此推测,增加植物体内的ABA、ZR的含量或降低IAA、GA₃的含量,都可以促进玫瑰的花芽分化;反之则抑制其花芽分化。     

Spatial Patterns of Seed Dispersal by White-Faced Capuchins in Costa Rica: Evaluating Distant-Dependent Seed Mortality

Spatial patterns of seed dispersal are the focus of numerous theoretical examinations of endozoochory. Here, we examine the spatial pattern of seed dispersal by white-faced capuchin monkeys Cebus capucinus in Santa Rosa National Park, Costa Rica, and the neighborhood characteristics and distance variables most closely associated with seed survival and germination, and seedling survival and growth in various locations. Overall, distance to the nearest fruiting conspecific tree has the most positive, consistent effect on growth and survival variables, which supports a variation of the Janzen–Connell seed escape hypothesis.     

镧和钐对月季切花衰老的影响

0.5×10-3mol·L-1的La3+和Sm3+溶液均能改善月季切花体内的水分平衡,增加切花鲜重和维持花径大小,减少花瓣溶质外渗,维持细胞膜结构的稳定性,降低呼吸速率,使切花瓶插寿命延长2￣3d。     

云南悬钩子蔷薇NBS-LRR类抗病基因同源克隆与分析

为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBSLRR序列中的保守区域设计简并引物,利用RTPCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBSLRR保守结构域的抗病基因同源序列（RGAs）,分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。