Regular Plasmid妹麗 Promoter Testing Vector (喝事for In Vivo Promoter Testing)

概述

This vector syste城場m is designed for ef資森ficient analysis of mammali為分an promoters in mouse models.&n開海bsp;Typically, a pu業件tative promoter of i冷綠nterest is cloned into this vector, u電算pstream of a LacZ rep問樹orter gene and th志城e resulting constr多唱uct is used to make transgen土錢ic mice. Express了費ion of the LacZ reporte這都r in transgenic embryos or adult 技國mice can then be u家用sed as a readout of promoter ac文刀tivity.

This vector system is u弟小seful for identifying prom鐘報oter elements, determining tiss機可ue-specificity of白年 promoters, comparing p請店romoter variants, line是睡age-tracing and m林動any other applications.

For further information abou會業t this vector syste林睡m, please refer to the papers習錢 below.

References	Topic
Comput Chem. 23:191 (1999)	Review on eukary市風otic promoter prediction雜車
J Biol Chem. 273:1河唱0530 (1998)	Analysis of promoter 說數activity in vivo using時光 a lacZ reporter plasmi厭是d

亮點

Our vector is based on 木數a regular plasmid system. The putati請說ve promoter to be上技 tested is placed i區遠mmediately upstream of a LacZ reporter歌廠 gene. While an active promo窗子ter would drive the expression很醫 of the downstream LacZ gene,多店 in the absence of promoter activity下中 there will little o年遠r no LacZ expression. LacZ is 火文used as the rep黑東orter because colorimetric林姐 staining of LacZ by X-gal in 作視whole-mount embryos o間東r tissue sections al商木lows highly sensitive det下熱ection of promoter河聽 activity in situ.

優勢

Easy generation of transgenic a能北nimals: The construct ca刀窗n be readily used to make tra輛黑nsgenic embryos or live mice with h吃這igh efficiency by章生 conventional pronuclear吧花 injection.

Simple and sensitive r訊坐eadout: When using LacZ as the report美風er, X-gal staining pr間木oduces a vivid blue product that is re筆匠adily detectible even at low expression笑坐 levels, resulting in very se朋樹nsitive readout o區家f promoter activi音森ty.

Technical simplicity: Delivering plasm厭紙id vectors into cells by conventio好理nal transfection or injection拍明 is technically straightfo樹身rward, and far easier than virus-員計based vectors which require the packa這公ging of live virus.

Very large cargo spac遠但e: Our vector can accommodate ~村姐30 kb of total DNA. This allow事舊s testing of lar的舞ge putative promoter seq月南uences.

不足之處

Random integration into the host 鄉如genome: When the vector is used to 計媽make transgenic mice 章離by pronuclear i和光njection, one or視嗎 more copies of the事熱 vector can integ文都rate randomly in 兵鄉the host genome. Neighboring genomi農樂c sequence at the坐間 integration site, coupled wit藍鐵h copy number variation麗農 and varying degree理現s of chewing back of the int綠動egrated fragment了科, could influence the level and spec暗妹ificity of reporter gene express些很ion. To overcome愛什 this, multiple transgenic lines are 用器generally needed for a given construc畫西t, so as to identify the common expres也照sion pattern shared among the multipl人業e lines, which is likely t黑個o be the true pa坐個ttern rendered by t麗畫he promoter.

載體關鍵元件

Promoter: Your promoter of inter城年est is placed he山放re.

LacZ: The beta-galactosidase 答錢reporter gene. The encoded資跳 enzyme converts the colorless and s吧理oluble X-gal to an inten哥學sely blue insoluble內森 product that stains 和開the cells in which 車低LacZ is expressed.

SV40 early pA: Simian virus 40 early polyadeny遠知lation signal. It faci關關litates transcriptional termination o海我f the upstream ORF.

Ampicillin: Ampicillin resistance gene. 麗討It allows the plasmid t知化o be maintained by ampicillin selectio線暗n in E. coli.

pUC ori: pUC origin of replication. Pla鄉用smids carrying this origin e小睡xist in high copy numbers in E. co就坐li.

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Regular Plasmid妹麗 Promoter Testing Vector (喝事for In Vivo Promoter Testing)