ProductOverview
Sonichedgehog(SHH)isusedtostudydifferentiationandexpansionofhumanandmouseembryonicandadultstemcells.StudiessuggestSHHisinvolvedinregulatingstemcellfatesofneuralandhematopoeticlineages1.Itcontrolscelldivisionofadultstemcellsandhasbeenimplicatedindevelopmentofsomecancers2.SHHisinstrumentalinearlyembryopatterninganddevelopment.Ithasbeenimplicatedasthekeyinductivesignalinpatterningoftheventralneuraltube,theanterior-posteriorlimbaxis,andtheventralsomites3.ThematureBIOLOGicallyactiveformofSHHmoleculeisproducedbyautocatalyticcleavageofitsprecursorproteinandcorrespondstoapproximatelytheN-terminalhalfoftheprecursormolecule.SonicHedgehogisanapproximately20kDaproteinexpressedinE.coliconsistingof175aminoacidresidues,includinganN-terminalIle-IlesequencesubstitutedforthenaturallyoccurringchemicallymodifiedCysresidue.
ProductSpecifications
Size
25µg
Source
E.coli
Formulation
Lyophilizedfroma0.22sterile-filteredsolutionin1xPBS &0.1MNaCl.
AminoAcidSequence
IIGPGRGFGKRRHPKKLTPLAYKQFIPNVAEKTLGASGRYEGKITRNSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDKLNALAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESKAHIHCSVKAENSVAAKSGG
UniprotAccessionNumber
Q15465,residues24-197
Purity
Greaterthan98%bySDS-PAGEanalysis
EndotoxinLevel
Lessthan0.1ng/µg(1EU/µg)ofSHHasdeterminedbytheLALmethod.
BiologicActivity
TheED50islessthan1μg/mlasdeterminedbyitsABIlitytoinducealkalinephosphataseproductionbyC3H10T1/2fibroblasts.
StorageandStability
StemfactorSHHisshippedatroomtemperature.LyophilizedSHHisstableforupto6monthsfromdateofreceiptwhenstoredat-20°Cto-80°C.ReconstitutedSHH,atconcentrationsgreaterthan0.1mg/ml,isstableforupto3monthswhenstoredat-20°Candfor1yearwhenstoredat-80°C.
Reconstitution
Centrifugebrieflyandthenreconstitutein1XPBStoyieldastocksolutionofnolessthan0.1mg/mlofSHH.Reconstitutedproteinshouldbeusedimmediatelyorstoredinworkingaliquotsat-20°C.Forlong-termstorage,itisrecommendedtoaddacarrierprotein(0.1%HSAorBSA).Avoidfreeze-thawcyclesasitcanresultinlossofactivity.
SpecificationSheets
- 03-0008ProductSpecificationSheet
SafetyDataSheets
- 03-0008SafetyDataSheet
References
- Briscoe,J.(2009)Makingagrade:SonicHedgehogsignalingandthecontrolofneuralcellfate.EMBOJ.28:457-465.
- Gough,N.M.,Gearing,D.P.,King,J.A.,Willson,T.A.,Hilton,D.J.,Nicola,N.A.,andMetcalf,D.(1988)Molecularcloningandexpressionofthehumanhomologueofthemurinegeneencodingmyeloidleukemia-inhibitoryfactor.Proc.Natl.Acad.Sci.USA85:2623-2627.
- Ingham,P.W.,andMcMahon,A.P.(2001)Hedgehogsignalinginanimaldevelopment:parADIgmsandprinciples.GenesDev15:3059-3087.
Stemgent mRNA体细胞重编程系统
产品特点
1.快速 Fast
Stemgent® MicroRNA-Enhanced mRNA Reprogramming System只需2周即可将靶细胞重编程为iPS细胞，与仙台病毒（Sendai Virus）、非整合型DNA载体（Episomal DNA Vectors）等传统iPS重编程方法相比，缩短实验周期超过50%以上（Figure. 1）。
Figure 1. Stemgent® mRNA重编程系统、仙台病毒依赖的重编程系统以及游离型DNA载体依赖的重编程系统重编程时程比较。
简便 Easy
与病毒、DNA载体依赖的iPS重编程手段不同，使用Stemgent® MicroRNA-Enhanced mRNA Reprogramming System对靶细胞进行重编程，生成的iPS细胞无需进行复杂耗时的下游筛选程序，只需简简单单6个步骤，即可轻松构建iPS细胞系。
| Description | Day | |
| Step 1 | Material Preparation | Prior to starting |
| Step 2 | Plate Cells | 0 |
| Step 3 | Transfect Cells | 1 to 12 |
| Step 4 | Identify Cells | 13-14 |
| Step 5 | Pick and Passage iPS Cell Colonies | 15-16 |
| Step 6 | Maintain iPS Cell Cultures | 16+ |
注：因所用细胞类型及实验条件不同，上述时间轴可能略有不同，仅供参考。
Figure 2. Stemgent® MicroRNA-Enhanced mRNA Reprogramming System重编程过程时间表、显微镜下观察到的重编程过程中靶细胞形态变化。第12天，形成的iPS克隆表达多能型Marker TRA-1-81。
高效 Efficient
病毒、DNA载体等依赖的iPS重编程技术，其重编程效率介于0.00001%到1%之间不等，而Stemgent® MicroRNA-Enhanced mRNA Reprogramming System的重编程效率远远大于1%，极大地提高了iPS克隆的产出率。
表1. 不同类型iPS重编程手段效率比较
| Reprogramming Method | Efficiency | Integrating | Screening |
| RNA | >1% | No | No |
| 仙台病毒（Sendai virus） | 0.01-1% | No | Yes |
| DNA载体（Episomal/Minicircle） | 0.00% | Possible | Yes |
| 慢病毒（Lentivirus） | 0.001-0.01% | Yes | Yes |
| 腺病毒（Adenovirus） | 0.0001-0.001% | Possible | Yes |
| Protein | 0.00% | No | No |
安全 Safe
某些iPS研究领域，例如iPS临床研究、治疗应用等，对iPS细胞的安全性提出了极高的要求。使用Stemgent® MicroRNA-Enhanced mRNA Reprogramming System重编程靶细胞，无需担心构建的iPS细胞系可能含有病毒载体残留，亦无需担心无关的基因整合进宿主基因组，是目前适用于临床研究及治疗的最理想选择。
而以仙台病毒（Sendai Virus）为载体的iPS重编程手段，其病毒基因组虽然不会整合进宿主染色体，仙台病毒本身也不具备复制能力，但是为了构建真正意义上的virus-free的iPS细胞系，通常需要经过一个10-20代、耗时费力的筛选过程，以确保最终得到的iPS细胞系无病毒载体残留。以慢病毒（Lentivirus）体的iPS重编程手段，病毒基因组能够永久整合进宿主基因组，会给后续的iPS细胞分析带来干扰。
高性价比 Cost Effective
以仙台病毒为载体的iPS重编程试剂盒非常昂贵，综合重编程效率、时间及劳动力成本、安全性等多重考虑，Stemgent® MicroRNA-Enhanced mRNA Reprogramming System无疑是性价比的上乘之选。
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