Copy and paste this code in Notepad@echo offtitle Connecting.....echo.set count=0:1if "%count%"=="300" goto 2set /a count+=1goto 1:2echo Wset count=0:3if "%count%"=="100" goto 4set /a count+=1goto 3:4clsecho.echo Waset count=0:5if "%count%"=="100" goto 6set /a count+=1goto 5:6clsecho.echo Wakset count=0:7if "%count%"=="100" goto 8set /a count+=1goto 7:8clsecho.echo Wakeset count=0:9if "%count%"=="100" goto 10set /a count+=1goto 9:10clsecho.echo Wake uset count=0:11if "%count%"=="100" goto 12set /a count+=1goto 11:12clsecho.echo Wake upset count=0:13if "%count%"=="100" goto 14set /a count+=1goto 13:14clsecho.echo Wake up,set count=0:15if "%count%"=="100" goto 16set /a count+=1goto 15:16clsecho.echo Wake up, Nset count=0:17if "%count%"=="100" goto 18set /a count+=1goto 17:18clsecho.echo Wake up, Neset count=0:19if "%count%"=="100" goto 20set /a count+=1goto 19:20clsecho.echo Wake up, Neoset count=0:21if "%count%"=="100" goto 22set /a count+=1goto 21:22clsecho.echo Wake up, Neo.set count=0:23if "%count%"=="100" goto 24set /a count+=1goto 23:24clsecho.echo Wake up, Neo..set count=0:25if "%count%"=="100" goto 26set /a count+=1goto 25:26clsecho.echo Wake up, Neo...set count=0:27if "%count%"=="800" goto 28set /a count+=1goto 27:28clsecho.color 0aecho Tset count=0:29if "%count%"=="50" goto 30set /a count+=1goto 29:30clsecho.echo Thset count=0:31if "%count%"=="50" goto 32set /a count+=1goto 31:32clsecho.echo Theset count=0:31if "%count%"=="50" goto 32set /a count+=1goto 31:32clsecho.echo The Mset count=0:33if "%count%"=="50" goto 34set /a count+=1goto 33:34clsecho.echo The Maset count=0:35if "%count%"=="50" goto 36set /a count+=1goto 35:36clsecho.echo The Matset count=0:37if "%count%"=="50" goto 38set /a count+=1goto 37:38clsecho.echo The Matrset count=0:39if "%count%"=="50" goto 40set /a count+=1goto 39:40clsecho.echo The Matrset count=0:41if "%count%"=="50" goto 42set /a count+=1goto 41:42clsecho.echo The Matriset count=0:43if "%count%"=="50" goto 44set /a count+=1goto 43:44clsecho.echo The Matrixset count=0:45if "%count%"=="50" goto 46set /a count+=1goto 45:46clsecho.echo The Matrix hset count=0:47if "%count%"=="50" goto 48set /a count+=1goto 47:48clsecho.echo The Matrix haset count=0:49if "%count%"=="50" goto 50set /a count+=1goto 49:50clsecho.echo The Matrix hasset count=0:51if "%count%"=="50" goto 52set /a count+=1goto 51:52clsecho.echo The Matrix has yset count=0:53if "%count%"=="50" goto 54set /a count+=1goto 53:54clsecho.echo The Matrix has yoset count=0:55if "%count%"=="50" goto 56set /a count+=1goto 55:56clsecho.echo The Matrix has youset count=0:57if "%count%"=="50" goto 58set /a count+=1goto 57:58clsecho.echo The Matrix has you.set count=0:59if "%count%"=="50" goto 60set /a count+=1goto 59:60clsecho.echo The Matrix has you..set count=0:61if "%count%"=="50" goto 62set /a count+=1goto 61:62clsecho.echo The Matrix has you...set count=0:63if "%count%"=="500" goto 64set /a count+=1goto 63:64clsecho.echo Fset count=0:65if "%count%"=="50" goto 66set /a count+=1goto 65:66clsecho.echo Foset count=0:67if "%count%"=="50" goto 68set /a count+=1goto 67:68clsecho.echo Folset count=0:69if "%count%"=="50" goto 70set /a count+=1goto 69:70clsecho.echo Follset count=0:71if "%count%"=="50" goto 72set /a count+=1goto 71:72clsecho.echo Folloset count=0:71if "%count%"=="50" goto 72set /a count+=1goto 71:72clsecho.echo Followset count=0:73if "%count%"=="50" goto 74set /a count+=1goto 73:74clsecho.echo Follow tset count=0:75if "%count%"=="50" goto 76set /a count+=1goto 75:76clsecho.echo Follow thset count=0:77if "%count%"=="50" goto 78set /a count+=1goto 77:78clsecho.echo Follow theset count=0:79if "%count%"=="50" goto 80set /a count+=1goto 79:80clsecho.echo Follow the wset count=0:81if "%count%"=="50" goto 82set /a count+=1goto 81:82clsecho.echo Follow the whset count=0:83if "%count%"=="50" goto 84set /a count+=1goto 83:84clsecho.echo Follow the whiset count=0:85if "%count%"=="50" goto 86set /a count+=1goto 85:86clsecho.echo Follow the whitset count=0:87if "%count%"=="50" goto 88set /a count+=1goto 87:88clsecho.echo Follow the whiteset count=0:89if "%count%"=="50" goto 90set /a count+=1goto 89:90clsecho.echo Follow the white rset count=0:91if "%count%"=="50" goto 92set /a count+=1goto 91:92clsecho.echo Follow the white raset count=0:93if "%count%"=="50" goto 94set /a count+=1goto 93:94clsecho.echo Follow the white rabset count=0:95if "%count%"=="50" goto 96set /a count+=1goto 95:96clsecho.echo Follow the white rabbset count=0:97if "%count%"=="50" goto 98set /a count+=1goto 97:98clsecho.echo Follow the white rabbiset count=0:99if "%count%"=="50" goto 100set /a count+=1goto 99:100clsecho.echo Follow the white rabbitset count=0:101if "%count%"=="50" goto 102set /a count+=1goto 101:102clsecho.echo Follow the white rabbit.set count=0:103if "%count%"=="400" goto 104set /a count+=1goto 103:104clsecho.echo Knock, knock, Neo.set count=0:105if "%count%"=="600" goto 106set /a count+=1goto 105:106exitSave it as anything.bat and run it....
Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.
Knock knock, Neo.
To achieve CRISPR/Cas9-mediated knock-in into Rosa26, we selected sgRNA target sequences spanning the XbaI site within the first intron, adapted to the homology regions of gene targeting vectors used for ES cells that cover sequences up- and downstream of this site [3]. As we have shown previously, sgRosa26-1 (Fig. 1a) exhibits high activity in mouse cells [13]. We therefore selected sgRosa26-1, together with a Cas9 mRNA that includes a plasmid coded polyadenine (polyA) tail (Cas9-162A) [14], for targeting in zygotes. The most effective concentrations of Cas9-162A and sgRosa26-1 RNAs were determined by microinjection of varying amounts of RNA into the pronuclei of C57BL/6 zygotes, followed by embryo culture to the blastocyst stage. Genomic DNA was extracted from each blastocyst and used for PCR amplification of the target region (Fig. 1b). PCR products were analyzed for NHEJ repair-associated deletions by digestion with XbaI or the T7 endonuclease I (T7EI). At the lowest concentrations of Cas9-162A (5 ng/μl) and sgRosa26-1 (2.5 ng/μl) RNAs, Rosa26 alleles from 40 % of the embryos exhibited sequence deletions, as shown by the presence of XbaI resistant bands, whereas T7EI assays were less sensitive (Fig. 1c). Sequencing of cloned PCR products from four blastocysts confirmed the presence of small deletions at the expected cleavage site. Of note, individual deletion events could generate new XbaI sites, causing an underestimation of gene editing events by XbaI digestion (Fig. 1d). Upon RNA microinjection of Cas9-162A at 25 ng/μl and sgRosa26-1 at 12.5 ng/μl, 80 % of cultured embryos showed XbaI resistant PCR products, a percentage that was not further increased at higher concentrations (Fig. 1e, Additional file 1: Figure S1). XbaI resistant PCR products represented a minor fraction in most of the samples, indicating the preferential modification of the Rosa26 allele in a heterozygous and/or mosaic pattern, although 10 % of the embryos showed processing of both alleles. We reasoned that conditions leading to Rosa26 deletions in the majority of embryos may also support knock-in events in at least a fraction of embryos, since HDR can occur in mammalian cells at 10 % of nuclease induced DSBs [15]. 2ff7e9595c
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