Expression and purification of biologically active recombinant quail stem cell factor in E. coli

Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. Previous studies have demonstrated that avian SCF is a requirement for the proliferation and survival of various cell types in vivo and in vitro. In the current study, recombinant quail stem cell factor was produced in Escherichia coli using a prokaryotic expression system. SCF was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by affinity chromatography on the Ni-NTA column. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Western blot analysis with the monoclonal antibody to the histidine tag identified SCF in the induced cell lysates and the purified sample. The recombinant SCF was approximately 22-23 kD in size. This protein was generated devoid of the signal peptide, the transmembrane domain, and the intracellular domain and, hence, resembles the soluble form of SCF. Biological activity was assayed using the in vitro survival of E12 chicken dorsal root ganglion-derived sensory neurons. The addition of recombinant quail SCF improved neuronal survival. Survival (20.6%) was the highest at the 50 ng/ml concentration of SCF. The availability of quail SCF will be a valuable tool to further resolve the function of stem cell factor in birds.     

Suppressors, Screens, and Genes: An Educational Primer for Use with "A Network of Genes Antagonistic to the LIN-35 Retinoblastoma Protein of Caenorhabditis elegans"

An article by Polley and Fay in this issue of GENETICS provides an excellent opportunity to introduce or reinforce concepts of reverse genetics and RNA interference, suppressor screens, synthetic phenotypes, and phenocopy. Necessary background, explanations of these concepts, and a sample approach to classroom use of the original article, including discussion questions, are provided.     

基于地高辛标记对小麦进行Southern杂交分析主要影响因素的优化和验证

以转基因小麦和野生型小麦DNA为材料,对利用地高辛标记对小麦基因组DNA进行Southern杂交分析的影响因素进行了优化研究,包括探针制备与纯化、样品DNA量、酶切体系、真空转印条件、杂交条件、免疫检测方法等。结果表明,对随机引物标记的模板和标记后的探针进行纯化可明显提高探针的标记效率,10μg高质量的DNA样品在80μl的体系中,酶切8～12h可获得良好的效果;真空转膜时使用碱性液比中性液获得的转膜效果更干净;试剂纯度、杂交温度及杂交炉转速等均对杂交效果产生重要影响;配合改进的CSPD涂布方法,使用化学发光检测系统比单纯使用X光片显像更易操作,背景更干净;本研究所优化的地高辛标记的小麦Southern杂交分析显示出较高的灵敏度和信噪比,结果稳定,可克服同位素标记对实验条件、设备及实验人员身体状况等限制,在普通实验室推广应用。     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

Isolation and characterization of 100 polymorphic microsatellite loci for the Siberian jay (Perisoreus infaustus)

We describe primers and polymerase chain reaction conditions to amplify 100 microsatellite loci from the Siberian jay (Perisoreus infaustus). The primers were tested on two geographically separated Finnish populations. The developed primer pairs yielded an average of 4.72 alleles per locus (range one to 17) and an average observed heterozygosity of 0.55 (range 0.04 to 1).     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

PERMANENT GENETIC RESOURCES: Twelve new microsatellite markers for the Chinese shrimp Fenneropenaeus chinensis

Twelve new microsatellite markers were isolated by sequencing random clones from a genomic library of Fenneropenaeus chinensis. The number of alleles per locus ranged from five to 15. The polymorphism information content ranged from 0.568 to 0.898, and observed and expected heterozygosities from 0.344 to 0.882 and from 0.691 to 0.915, respectively. All loci except one conformed to Hardy-Weinberg equilibrium. These markers, described here for Chinese shrimp, will be further used to analyse the species' population genetics.     

Chemical gene synthesis: strategies, softwares, error corrections, and applications

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene structure, expression and function. Modified genes and consequently protein/enzymes can bridge genomics and proteomics research or facilitate commercial applications of gene and protein technologies. In this review, we will summarize various strategies, designing softwares and error correction methods for chemical gene synthesis, particularly for the synthesis and assembly of long DNA molecules based on polymerase cycling assembly. Also, we will briefly discuss some of the major applications of chemical synthesis of DNA sequences in basic research and applied areas.     

M-PCR： a powerful method for rapid molecular identification of Trichogramma wasps （Hymenoptera： Trichogrammatidae）

Two species-specific primers were designed depending on ITS2 sequence variation of 37 Trichogramma wasps, and these primers were applied to establish an assay,multiplex PCR (M-PCR), for molecular diagnosis of two important Trichogramma wasps,T. confusum and T. dendrolimi, in China. Multiplex-PCR results showed that only target species produced two PCR products, one product of ITS2 region species-specific amplification and one product of its ITS 1 region universal amplification, but other species produced only one ITS1 universal PCR product. Using this method, the target Trichogramma species can be distinguished from other Trichogramma species. Molecular identification based on M-PCR has particular value over morphological technology and other approaches, such as normal molecular and biochemical methods. Furthermore, because M-PCR assay can avoid false negative results, which frequently happen in PCR reaction, this method will be much more accurate and useful for Trichogramma identification, and can be developed as an easy and rapid diagnostic kit applied in the identification and quality monitoring of Trichogramma mass products both in the factory and in the field. Such an easy and rapid diagnostic kit will be valuable in the application of Trichogramma species as a biological control.