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載體構(gòu)建 
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哺乳動物基因表達載體

概述

在生物醫(yī)學(xué)領(lǐng)域,利用常規(guī)轉(zhuǎn)染將質(zhì)粒載體轉(zhuǎn)到哺乳動物細(xì)胞中是使用最廣泛的方法。雖然近年來開發(fā)了不少更為先進的基因?qū)胼d體系統(tǒng)(如慢病毒載體、腺病毒載體、AAV載體和piggyBac等),但常規(guī)質(zhì)?;虮磉_載體仍被大多數(shù)實驗室使用,這主要得益于其在技術(shù)上的簡便性以及在各種類型細(xì)胞中的高效轉(zhuǎn)染率。常規(guī)質(zhì)粒載體轉(zhuǎn)染的關(guān)鍵特點是短暫性,并且在宿主基因組中的整合效率非常低(通常小于1%)。

關(guān)于常規(guī)質(zhì)?;虮磉_載體的更多信息,請參考以下文獻。

參考文獻主題
Mol Biotechnol. 16:151 (2000)Overview of vector design for mammalian gene expression
EMBO J. 12:2539 (1993)Transcription blocker prevent transcriptional interference

亮點

我們的載體系統(tǒng)已經(jīng)過優(yōu)化,其在大腸桿菌中具有高拷貝復(fù)制能力,且對細(xì)胞具有高效轉(zhuǎn)染率。根據(jù)用戶選擇的篩選標(biāo)記基因,可對轉(zhuǎn)染了該載體的細(xì)胞進行篩選或者可視化追蹤。

優(yōu)勢

技術(shù)簡單:常規(guī)轉(zhuǎn)染即可把質(zhì)粒轉(zhuǎn)入細(xì)胞,相比起病毒載體需要進行病毒包裝,過程更簡單。

載體容量非常大:我們的載體總?cè)萘靠蛇_30 kb,其中質(zhì)粒骨架只占3 kb,有足夠大的容量可以放置客戶所感興趣的序列。

高水平表達:常規(guī)質(zhì)粒轉(zhuǎn)染細(xì)胞后,可以產(chǎn)生非常高的拷貝數(shù)(每個細(xì)胞多達幾千拷貝),使外源基因能高水平表達。

不足之處

載體DNA非整合型:常規(guī)質(zhì)粒在細(xì)胞里主要以游離DNA狀態(tài)存在,所以常規(guī)質(zhì)粒轉(zhuǎn)染也稱為瞬時轉(zhuǎn)染。然而,質(zhì)粒DNA也可以以非常低的概率永久整合到宿主基因組中(質(zhì)粒轉(zhuǎn)染每102~106個細(xì)胞可能會有一個細(xì)胞的基因組被整合,整合效率取決于細(xì)胞類型)。如果將攜帶了藥物抗性基因或者熒光標(biāo)記基因的載體轉(zhuǎn)到細(xì)胞中,在經(jīng)過擴大和傳代培養(yǎng)后,可以使用藥物篩選或細(xì)胞分選來獲得穩(wěn)定整合了該載體的細(xì)胞。

可轉(zhuǎn)染的細(xì)胞類型受限:不同類型細(xì)胞的質(zhì)粒轉(zhuǎn)染效率差異很大。非分裂細(xì)胞比分裂細(xì)胞更難轉(zhuǎn)染,原代細(xì)胞比永生化細(xì)胞系更難轉(zhuǎn)染。一些重要的細(xì)胞類型更是難以轉(zhuǎn)染,如神經(jīng)元和胰島β細(xì)胞。另外,質(zhì)粒轉(zhuǎn)染局限于體外細(xì)胞實驗,很少運用到活體實驗。

基因拷貝數(shù)在不同細(xì)胞中呈現(xiàn)差異性:盡管成功的轉(zhuǎn)染可以在單個細(xì)胞里產(chǎn)生非常高的拷貝數(shù),但不同細(xì)胞間卻有高度的差異性。在一些細(xì)胞中,質(zhì)粒拷貝數(shù)非常高,而在另外一些細(xì)胞卻是低拷貝甚至沒有。而病毒轉(zhuǎn)導(dǎo)更傾向于相對均勻地把基因轉(zhuǎn)到細(xì)胞中。

載體關(guān)鍵元件

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Representative vector design
VB IDVector nameDescriptions
VB010000-9486reypRP[Exp]-CMV>EGFP/PuroA regular plasmid encoding an EGFP: puromycin resistance fusion protein driven by a CMV promoter.
VB900120-0467nufpRP[Exp]-CAG>DD-CreA regular plasmid encoding a destabilized Cre recombinase protein, which functions in the presence of trimethoprim (TMP), under the control of a CAG promoter.
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