Our pUASTB vector 計鐵system is highly effectiv近空e for generating transge裡喝nic flies and controlling transgene兵民 expression. This system拍黃 has the capability to utili短報ze either the Dros還煙ophila P-element tran跳上sposon system (like pUAST) or the ba議坐cteriophage φC3跳的1 integration s劇要ystem (like pUASTattB), and can be u訊文sed for achieving ubiqu費家itous, tissue-specific or 妹還inducible transgene expressio工員n. Thus, the pUASTB vector sy裡內stem can be used for either P t店懂ransposon-mediated or 少制φC31 integrase-mediated道商 insertion of your gene of interest. 冷們In either case, the user selec什通ted gene of interest is cloned into 費術pUASTB in a region brac秒跳keted by two P-element ter笑鐘minal repeats and near間西 an attB recombination si街秒te.

To utilize P tra林美nsposon-mediated insert請子ion, the pUASTB plasmid and a P t我用ransposase-expressing helper玩電 plasmid are co-introduce西訊d into host cells or embryos. As a r要內esult, the transposase produced f兵還rom the helper plasmid re照新cognizes the two P-e見兒lement terminal repeats on the pUASTB p畫分lasmid, and inserts the flanked region物低 including the two P-element t秒不erminal repeats 制動into the host genome. P transposase-月遠mediated insertion oc北子curs without any significant 還風bias with respect 放雜to insertion site sequence.

To utilize φC31 integrase-mediated ins路嗎ertion, the pUASTB plasmid and 那是a φC31 integrase-還校expressing helper plasmid are co-嗎他introduced into host cells or 制暗embryos containing attP landing sit化校es. The φC31 integrase mediates ir友看reversible recombination between attB的件 and attP sites, re遠訊sulting in the linearization an錯子d integration of the pUASTB vector i道很nto the host genome.

The mini white gene on水站 the pUASTB vector encodes for eye c能很olor and acts as a marker for the iden雨近tification of transgeni路城c flies which have unde吧冷rgone successful genetic recombination員唱 of the transgen筆他e.

The user-defined promoter versi弟男on of the pUASTB Drosophi些農la gene expression vector allow行地s users to select a promoter of冷開 their choice from our Drosophila見海 promoter database for driving the e紅知xpression of their 妹我gene of interest (GOI) depen用西ding upon their experimental goal行章. Our Drosophila promoter database offe船大rs the following promoter choices: u國機biquitous promoters i街相ncluding actin 5C, polyu長嗎biquitin and alpha-1 tubul問能in for driving ubiquitous 但事expression of the GOI; tissue-spe文錯cific promoters suc務下h as Rh2 for driving GOI expression, sp歌工ecifically in Drosophila ocelli an業還d inducible promoters間子 such as Mtn for achievi店我ng inducible expression of the GOI with風房 the presence of Gu+.

For further information ab作綠out this vector system, ple匠秒ase refer to the papers below.

Site-specific insertion: φC31-based insertion is sit錯火e-specific, gene可一rally occurring only at at商北tP sites, and not elsewhere in t醫土he genome. This說話 reduces the risk of disrup懂林ting endogenous ge話吧nes or having inserti一的on site position e哥樹ffects on transgene expre吃是ssion.

Flexibility: The user-defined prom動遠oter version of the pUASTB vector allow好紅s users to add a ub高鐘iquitous, tissue-spec說還ific or inducible promoter for dr也中iving their GOI depending upon t動相heir experimental goa很物l.

High efficiency: Achieving germ-line transge公費nesis using φC31 integrase vectors is放服 more efficient農劇 than P-element based vector sy近坐stems such as pUAST物喝.

Random genomic inse數間rtion: If P transposase-mediated insertion of少議 pUASTB is used, random integration of 國科P-elements can make it diffic很雜ult to map genomic ins文看ertion sites, and 了謝genomic position can affect transgene 分綠expression. Additionally, transgene ins愛放ertion into genes or regulatory 雨有elements within th讀男e genome can affe錢醫ct endogenous genes.

Technical complexity: The generation of transgenic Drosophila子離 requires embryonic inje計從ction and fly hu謝你sbandry, which can be technically路市 difficult. 

Requires attP insertion 長近sites: The use of φC31 integrase-mediated 水近insertion of pUASTB req家得uires the use of spe鐵資cialized host lines carrying attP “l錯議anding sites” in their gen討唱ome.

P-element 3’ end: Right termi作哥nal repeat, or 3' ter地男minal repeat, of the P element. Whe美員n a DNA sequence i河化s flanked by the 3’ and 5’ P-elem公讀ent terminal repeats, the P 老分transposase can recognize them and in亮討sert the flanked r術有egion into the host genome.

Promoter: The user-selected 這作promoter driving the down來來stream gene of 和兵interest is placed here.

Kozak: Drosophila Kozak consensus se票厭quence. It is placed in fron也高t of the start co路算don of the ORF of業聽 interest to facilitate translation i話一nitiation in eukaryotes.

ORF: The open reading frame 不見of your gene of interest is plac老飛ed here.

SV40 terminator: Simian virus 40 transcriptio匠媽nal terminator. Co家子ntains the SV40 small T intron and 相歌the SV40 early 師河polyadenylation厭書 signal.

attB site: The bacterial att筆做achment site, attB, recognized by 又很the bacteriophage φC31 ser黑我ine integrase. φC31 inte坐兵grase can cataly爸開ze site-specific integration of att工開B-containing plasm林熱ids into attP-containing docki雜數ng or landing si影火tes that have been introduced into議還 host genomes.

mini-white: A variant of the Drosophi議我la white gene. The mini-white gene i是北s a dominant marker for a吃在dult fruit fly eye color,錢開 which can be used as a reporter to舞化 identify trans和船genic events in a whi妹鄉te mutant background.

P-element 5’ end:&化得nbsp;Left terminal repeat, or 5' term白中inal repeat, of the P element. W快南hen a DNA sequence i少嗎s flanked by the 3’ and 5’ P-element t和工erminal repeats, the P tra風鄉nsposase can recogn呢自ize them and insert 農妹the flanked region into the host gen日間ome.

pUC ori: pUC origin of replica件人tion. Plasmids carrying this orig爸嗎in exist in high copy numbers in E. col間聽i.

Ampicillin: Ampicillin resista高車nce gene. It allows the p區黃lasmid to be maintained by ampicil通不lin selection in E. coli.