"Kev loj hlob ntawm cov neeg tshiab ic3a / CAS12A-Foliadiated"Ib-lauj kaub" dual Kev tshawb nrhiav rau cov kab mob hloov kho
Kev siv caj dab hloov caj dab tau dav thoob ntiaj teb, thiab ntau lub tebchaws tau txhim kho lawv cov kev tswj hwm kev nyab xeeb hloov cov qoob loo. Kev loj hlob sai thiab raug rau cov txheej txheem caj ces yog qhov tseem ceeb heev rau kev txhawj xeeb txog kev hloov pauv pej xeem txog cov khoom hloov pauv.
Hauv txoj kev tshawb fawb no, cov pab pawg tshawb fawb tau tsim lub zog tshiab ntawm camv35s thiab nos kuaj pom kev hloov kho ntawm cov kab mob caj ces.
Kev sim ua
Cov khoom lag luam dab tsi yog tshuaj ntsuam los ntawm Cas13a thiab Cas12a thiab tom qab ntawd Fluorescent ntsuas lossis los ntawm kev ntsuas fluorication nrog ob t kab kom ua tiav dual-channel nyeem ntawv. Tag nrho cov txheej txheem tiav nyob rau hauv tsawg dua 35 feeb, nrog kev kuaj pom kom tsawg li 20 daim ntawv luam.
(Txoj kev cai ntawm MR-DCA nrhiav tau ntawm camv35s thiab nos)
Tag nrho cov kev sim tau muab faib ua peb qib: Mira Amplification, cov neeg raug tua, thiab ob txoj kev ntawm kev txhais.
Camv35s Gene raug siv los ua lub hom phiaj ntawm CAS13A, thiab Nos gene yog siv los ua Cas12a. Ob khub ntawm MMA Primers tau tsim feem. Cov txheej txheem CAS13A tsuas yog paub txog RNA, yog li cov chaw t7 rau pem hauv ntej yog tsim, uas muaj tus neeg xa khoom T7 thiab ua kom ua haujlwm tom ntej. Ib qho PAM tau ntxiv rau tus nos gene. Qhov saum toj no ntxiv kom ntseeg tau tias ob qho cas ntawm 13a thiab cov protein tuaj yeem khi lawv cov kev cuam tshuam lossis kev cuam tshuam lossis hla kev tawm ntawm ob lub nruab nrog.
Sim teeb meem
Rhiab heev thiab muaj feem cuam
Cov qauv tau diluted nyob rau hauv ib tug gradient los sim cov fluorescence ntawm lub mira cov tshuaj tiv thaiv. Nws tau pom tias kev siv fluorescence tseem tuaj yeem kuaj pom ntawm qhov piv txwv txog 20 μL. Qhov kev xav ntawm 20 μl tau thaum kawg xaiv rau kev sim.
(Sib txawv cov qauv thiab kuaj cov hlua khi tau siv rau kev tsim cov xim thiab kev siv ntau yam kev suav)
Txhawm rau ntsuas qhov tshwj xeeb ntawm MR-DCA, cov pab pawg tshawb fawb tau sim sib xyaw cov tsiaj ntawm cov tsiaj uas tsis hloov chaw thiab tsis hloov chaw. Cov txiaj ntsig tau pom tias tsuas transgenic cov qauv muaj camv35s thiab nos muaj cov qauv fluorescence ntawm lwm cov qauv tau nyob ze ntawm qhov kev tswj hwm tsis zoo. MR-DCA tuaj yeem siv los ua ib qho tshwj xeeb dual-phiaj nucleic acid kuaj tau platform.
24 Qoob loo cov qauv raug sim los ntawm MR-DCA. Ntawm lawv, 1, 2, 15, 16, 21, thiab 22 yog transgenic pob kws muaj schv35s txhawb nqa. 7, 8, 23, thiab 24 yog tus kab mob-tiv taus transgenic mov uas muaj tus nos terminator. 3, 4, 11, 11, 13, 13, 14, 17, thiab 18 yog transgenic taum pauv uas muaj Camv35s txhawb nqa thiab nos tininator.
(Kev ntsuas cov lus ntawm kev kuaj xyuas ntawm 24 kuaj)
Kev tawm tsam sai
Lub sijhawm muaj qee qhov tseem ceeb hauv kev tshawb nrhiav hauv thaj chaw kom pom. Pab neeg tshawb fawb pom tias fluorescence cov cim ntawm kev tshaib fluorescence ntawm kev paub thaum lub sijhawm fluorescence thaum lub sijhawm tshuaj tiv thaiv yog 10 feeb. Mira ua ke nrog cov tshuaj tiv thaiv crispr kuj pom lub ntsej muag daj tom qab 15 feeb.
(Fluorescence teeb liab ntawm sib txawv Mira cov sij hawm)
(Cov xim kev tsim kho ntawm qhov kev xeem ntawm kev sib txawv Mira cov sij hawm thiab kev siv kom paub tseeb kev muab xam)
Sim sim
Qhov kev sim sim tau qhia tau hais tias tus me ntsis mira muaj qhov zoo tshwj xeeb thiab tuaj yeem muaj cov khoom lag luam zoo ntawm Camv35S thiab Nos. CAS13A thiab Cas12A lub tshuab tawm ntawm nws tus kheej tsis muaj kev cuam tshuam nrog txhua lwm yam. Txoj kev ntsuas MR-DCA tuaj yeem raug ntes 20 daim ntawv luam ntawm qhov kub ntawm 35 feeb, uas muaj txiaj ntsig zoo hauv kev ntes cov qoob loo hauv thaj teb.
Lus Qhia
Xov Xwm Neeg Sau Ntawv Npe: "Journal of Advanced tshawb fawb
Tshooj cai npe: "Kev loj los ntawm cov neeg tshiab ic3a / metriated" ib-lub lauj kaub "ob lub lauj kaub" dual kev tshawb pom ntawm kev hloov kho cov qoob loo
Cuam tshuam Factor: 11.4
Pab Pawg Tshawb Xyuas: Wang Xiabofu, Zhejiang Academy ntawm Kev Kawm Txuj Ci