PiggyBac Non-Coding RNA Expression 讀物Vector

The piggyBac non-coding RNA expressi文老on vector is a highly effective t家光ool for transfection-based permanent in報老tegration of non內廠-coding RNAs into the host genome o志時f mammalian cells. Non-co新去ding RNAs include a wide學能 variety of short (<30 nucleotides) 刀多and long (>200 姐刀nucleotides) functional RNA molec金月ules such as micro RNAs (miRNA什也s), small inter窗還fering RNAs (siRNAs師少), piwi-interact光城ing RNAs (piRNAs), small nuclear RN輛我As (snRNAs), small nucleolar 她她RNAs (snoRNAs), l地金arge intergenic non-codin店木g RNAs (lincRNAs), intronic long低市 non-coding RNAs (intronic lncRNAs購資), natural antisense transcripts (NA物水Ts), enhancer RNAs (eRNAs) 請又and promoter-associated RNAs (PARs)飛影, none of which are translated請飛 into proteins,體日 however have been found to 也術play important roles in many 了是cellular processes such as DN科關A replication, epigenetic小民 regulation, transcriptional an跳知d post-transcriptional r錢老egulation and translati鄉美on regulation.

The piggyBac non-codin議業g RNA expression vector uses an&n男些bsp;RNA polymerase II promo女對ter to drive the express麗雨ion of the user-sel黑子ected non-coding RN些家A gene. This allows t科亮he use of tissue西老-specific, inducibl門章e, or variable-stren愛習gth promoters, enabling a variet遠低y of experimental applications. For RN多鄉A polymerase II-mediated t兒東ranscription, the start s用算ite is typically in the 3' reg費唱ion of the promoter while th友司e termination site is wi文日thin the polyA signal sequence.城錯 As a result, th舞但e transcript generated from 老兵this vector does 體農not correspond precisely to th話內e selected non-codin為刀g RNA gene, but contains some addit很市ional sequences both upstream and d東木ownstream. 

The piggyBac system contai熱近ns two vectors, both engineere人海d as E. coli plasmids. One vector, 喝飛referred to as 要草the helper plasmid, encodes the tran老他sposase. The other vector, referred t物到o as the transposon pl放動asmid, contains two terminal repeats (T友能Rs) bracketing the regi動熱on to be transposed. The non-美紙coding RNA to be delivered into host雜人 cells along with the user-selecte關男d promoter is cloned into this region報林.

When the helper and transp花高oson plasmids are co-transfected水為 into target cells, the 他木transposase produced from the helpe議窗r would recognize the two TRs計森 on the transposon, and in他信sert the flanked regio亮術n including the two T線購Rs into the host g章路enome. Insertion兒飛 typically occurs at host chromosomal s文術ites that contain the TTAA sequence, w事一hich is duplicated 如業on the two flank你友s of the integra黃村ted fragment.

PiggyBac is a class II tran很如sposon, meaning that i謝很t moves in a cut-and-paste manne爸資r, hopping from plac木資e to place without leaving c文化opies behind. (In cont呢計rast, class I transposons move in a低了 copy-and-paste manner.) Becaus水為e the helper plasmid i但中s only transiently t雨西ransfected into hos秒動t cells, it will get亮放 lost over time. With the loss 嗎就of the helper plasmid麗是, the integration of the transposon i要就n the genome of hos讀地t cells becomes permane煙劇nt. If these cells are t東紙ransfected with the hel東森per plasmid again,坐聽 the transposon could get excise能到d from the genome of some cells, f自爸ootprint free.

For further information會到 about this vector system, ple車著ase refer to the papers below他舞.

The piggyBac non-coding RNA expression校鄉 vector along with the 師南helper plasmid are optimized for high c內又opy number replication in話做 E. coli, efficient 歌金transfection int白用o a wide range of target cell草如s, and high-level express錯呢ion of the transgene car章還ried on the vector.

Limited cell type 風工range: The delivery of pi區兵ggyBac vectors into cells reli亮視es on transfection. The effici拿區ency of transfection can vary greatly f說你rom cell type to cell type. N報門on-dividing cells are ofte喝裡n more difficult to transfect than div關生iding cells, and primary cells a了一re often harder to transfect要對 than immortalized cell lines. Some 員道important cell types, such 在又as neurons and 但都pancreatic β cells, ar白時e notoriously difficult to transfe村吃ct. Additionally, plasmid transfectio也來n is largely limited to in vitro app亮討lications and rarely used 務答in vivo. These issues limit t電自he use of the piggyBac s計對ystem.

5' ITR: 5' inverted terminal 公如repeat. When a DNA sequence 南舞is flanked by two ITRs, 冷站the piggyBac transpose can reco信國gnize them, and inse學都rt the flanked region inc費頻luding the two ITRs into the計了 host genome.

Promoter: The promoter driving your non-coding報村 RNA of interest is 遠舊placed here.

Non-coding RNA:農答 The non-coding RNA of業慢 your interest is placed匠時 here.

rBG pA: Rabbit β-globin poly個照adenylation signal時黃. It facilitates tr吃弟anscriptional terminatio體上n of the upstream non-coding RNA.

CMV promoter: Human cytomegalovi綠林rus immediate early promoter.中土 It drives the ubiquitous expression o動街f the downstream marker gene.

Marker: A drug selectio錯畫n gene (such as neomycin resistance),票工 a visually detecta笑快ble gene (such as EGFP快高), or a dual-reporter gene (such as輛民 EGFP/Neo). This allows cells transduc黃是ed with the vector to be selec雪妹ted and/or visuali通海zed.

BGH pA: Bovine grow水現th hormone poly什音adenylation signal. It 她又facilitates transcriptional termin女鐘ation of the upstream ORF可音.

3' ITR: 3' inverted terminal repe個好at.

Ampicillin: Ampicillin resistance 視我gene. It allows the plasmid制林 to be maintained by am一舊picillin selection in E. coli.

pUC ori: pUC origin of replicatio機讀n. Plasmids carrying this origin exist 麗城in high copy numbers in E.拍門 coli.