Enhancing the stability and ecological safety of mass‐reared transgenic strains for field release by redundant conditional lethality systems

The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically‐based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline‐suppressible (Tet‐off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline‐containing diet, but female‐lethal and male sterile in tetracycline‐free conditions. This would allow their most efficacious use in a unified system for sterile male‐only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet‐off lethal survival determinations, development of large‐scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet‐off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2¹ mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host‐specific cognates to be created for a wide range of insect species.     

Induced pluripotent stem cell‐derived conditional medium promotes Leydig cell anti‐apoptosis and proliferation via autophagy and Wnt/β‐catenin pathway

Leydig cell transplantation is a better alternative in the treatment of androgen‐deficient males. The main purpose of this study was to investigate the effects of induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) on the anti‐apoptosis, proliferation and function of immature Leydig cells (ILCs), and illuminate the underlying mechanisms. ILCs were exposed to 200 μmol/L hydrogen peroxide (H₂O₂) for 24 hours with or without iPS‐CM treatments. Cell apoptosis was detected by flow cytometric analysis. Cell proliferation was assessed using cell cycle assays and EdU staining. The steroidogenic enzyme expressions were quantified with Western blotting. The results showed that iPS‐CM significantly reduced H₂O₂‐induced ILC apoptosis through down‐regulation of autophagic and apoptotic proteins LC3‐I/II, Beclin‐1, P62, P53 and BAX as well as up‐regulation of BCL‐2, which could be inhibited by LY294002 (25 μmol/L). iPS‐CM could also promote ILC proliferation through up‐regulation of β‐catenin and its target proteins cyclin D1, c‐Myc and survivin, but was inhibited by XAV939 (10 μmol/L). The level of bFGF in iPS‐CM was higher than that of DMEM‐LG. Exogenous bFGF (20 ng/mL) or Wnt signalling agonist lithium chloride (LiCl) (20 mmol/L) added into DMEM‐LG could achieve the similar effects of iPS‐CM. Meanwhile, iPS‐CM could improve the medium testosterone levels and up‐regulation of LHCGR, SCARB1, STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B3 and SF‐1 in H₂O₂‐induced ILCs. In conclusion, iPS‐CM could reduce H₂O₂‐induced ILC apoptosis through the activation of autophagy, promote proliferation through up‐regulation of Wnt/β‐catenin pathway and enhance testosterone production through increasing steroidogenic enzyme expressions, which might be used in regenerative medicine for future.     

云南松胚性愈伤组织诱导及增殖

以云南松成熟合子胚为外植体,在DCR培养基上诱导胚性愈伤组织,探索最佳消毒剂用量及激素浓度配比。用不同的培养方法增殖胚性愈伤组织,并对胚性愈伤组织形成过程中形态学与细胞学变化进行观察。其后提高培养基中肌醇浓度,增大渗透压,添加ABA,诱导早期原胚。结果表明,经5%次氯酸钠消毒20 min,使用添加8mg/L 2,4-D1、mg/L 6-BA、500 mg/L CH以及500 mg/L谷氨酰胺的DCR培养基接种,诱导率较高,可达70%以上;采用在愈伤组织周围添加少量培养过该愈伤组织的培养基的方式继代,愈伤组织增殖率可由20%显著提高至50%左右。在增殖培养基中添加4~6 mg/L ABA后,胚性细胞可逐渐发育形成胚性胚柄团。     

Cre-mediated recombination in the skin melanocyte lineage

Organ-specific expression of a Cre recombinase allows the analysis of gene function in a particular tissue or cell type. Using a 6.1 kb promoter from the mouse tyrosinase gene, we generated and characterized two lines of transgenic mice that express Cre recombinase in melanoblasts. Utilizing a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts of the skin from embryonic day 11.5. In addition, Cre-expression was detected in the skin and eyes of mice. Cre transgene activity was occasionally detected in the brain and peripheral nerves but not in other tissues. When Tyr::Cre mice were crossed with mice carrying a homozygous loxP conditional mutation for the insulin-like growth factor receptor gene (Igf1r), Cre-melanoblast-specific recombination pattern was confirmed and no abnormal phenotype was observed. In conclusion, Tyr::Cre transgenic mice provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.     

Physiological dormancy in seeds of tropical montane woody species in Hawai`i

Worldwide, there is relatively little information on seed dormancy and germination of tropical montane species. Our aim was to help fill this knowledge gap by conducting seed dormancy/germination studies on woody species from this vegetation zone in Hawai`i. All species had water-permeable seeds with a fully developed embryo. Seeds of 29 species (23 genera) were incubated in light/dark at 15/6, 20/10 and 25/15°C and germination monitored at 2-week intervals for 16–128 weeks. Seeds of Chenopodium oahuense, Dubautia menziesii and Silene lanceolata were non-dormant (ND) and those of 26 other species had physiological dormancy (PD); 10 of the 26 species had conditional PD. The optimum germination temperature regime(s) was (were) 25/15°C, 17 species; 25/10 and 20/10°C, 2; 20/10°C, 6; 20/10 and 15/6°C, 2; and 15/6°C, 2. Worldwide, PD in the woody genera included in our study is more common than ND. In addition to its contribution to the world biogeography of seed dormancy/germination, this study will be useful to conservation biologists who need to germinate seeds of tropical montane species.     

Mx1‐Cre mediated Rgs12 conditional knockout mice exhibit increased bone mass phenotype

Regulators of G‐protein Signaling (Rgs) proteins are the members of a multigene family of GTPase‐accelerating proteins (GAP) for the Galpha subunit of heterotrimeric G‐proteins. Rgs proteins play critical roles in the regulation of G protein couple receptor (GPCR) signaling in normal physiology and human diseases such as cancer, heart diseases, and inflammation. Rgs12 is the largest protein of the Rgs protein family. Some in vitro studies have demonstrated that Rgs12 plays a critical role in regulating cell differentiation and migration; however its function and mechanism in vivo is largely unknown. Here, we generated a floxed Rgs12 allele (Rgs12^flox/flox) in which the exon 2, containing both PDZ and PTB_PID domains of Rgs12, was flanked with two loxp sites. By using the inducible Mx1‐cre and Poly I:C system to specifically delete Rgs12 at postnatal 10 days in interferon‐responsive cells including monocyte and macrophage cells, we found that Rgs12 mutant mice had growth retardation with the phenotype of increased bone mass. We further found that deletion of Rgs12 reduced osteoclast numbers and had no significant effect on osteoblast formation. Thus, Rgs12^flox/flox conditional mice provide a valuable tool for in vivo analysis of Rgs12 function and mechanism through time‐ and cell‐specific deletion of Rgs12. genesis 51:201–209, 2013. © 2013 Wiley Periodicals, Inc.     

The role of conservative versus innovative nesting behavior on the 25‐year population expansion of an avian predator

Species ranges often change in relation to multiple environmental and demographic factors. Innovative behaviors may affect these changes by facilitating the use of novel habitats, although this idea has been little explored. Here, we investigate the importance of behavior during range change, using a 25‐year population expansion of Bonelli's eagle in southern Portugal. This unique population is almost exclusively tree nesting, while all other populations in western Europe are predominantly cliff nesting. During 1991–2014, we surveyed nest sites and estimated the year when each breeding territory was established. We approximated the boundaries of 84 territories using Dirichlet tessellation and mapped topography, land cover, and the density of human infrastructures in buffers (250, 500, and 1,000 m) around nest and random sites. We then compared environmental conditions at matching nest and random sites within territories using conditional logistic regression, and used quantile regression to estimate trends in nesting habitats in relation to the year of territory establishment. Most nests (>85%, n = 197) were in eucalypts, maritime pines, and cork oaks. Nest sites were farther from the nests of neighboring territories than random points, and they were in areas with higher terrain roughness, lower cover by agricultural and built‐up areas, and lower road and powerline densities. Nesting habitat selection varied little with year of territory establishment, although nesting in eucalypts increased, while cliff nesting and cork oak nesting, and terrain roughness declined. Our results suggest that the observed expansion of Bonelli's eagles was facilitated by the tree nesting behavior, which allowed the colonization of areas without cliffs. However, all but a very few breeding pairs settled in habitats comparable to those of the initial population nucleus, suggesting that after an initial trigger possibly facilitated by tree nesting, the habitat selection remained largely conservative. Overall, our study supports recent calls to incorporate information on behavior for understanding and predicting species range shifts.     

Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology

Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx‐based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene‐trap mutagenesis. The SAGFLEx gene‐trap cassette comprises the rabbit β‐globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site‐specific recombinases Cre and Flp. Insertion of the gene‐trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene‐trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial‐ and temporal‐controlled manner. genesis 54:19–28, 2016. © 2015 Wiley Periodicals, Inc.