4 Material and Methods
4.1 Material
4.1.5 DNA oligonucleotides
DNA oligonucleotides were purchased lyophilized and salt-free from eurofins.com or biomers.net, resolved in TE buffer to a concentration of 100 µM and stored at -20 °C. The only exception is oligonucleotide Nr. 284, which was stored at -80 °C.
Table 14. List of oligonucleotides with sequence information.
No. Name Sequence Application
46 T7-Primer TAATACGACTCACTATAGGG Sequencing
91 M13fw GTAAAACGACGGCCAG Sequencing,
Genotyping of Hsp70l:Cyp26a1 92 hsp:cyp26a1-fw2 GAGCAAAGGTCTGGAGGA Genotyping of Hsp70l:Cyp26a1 183 ERT2-Gal4-EcoRV ACGTAGATATCGAATGGCCGGTGAC
ATGAG
Cloning of p380 184 ERT2-Gal4-NotI ATAGCGGCCGCTTAGTTACCCGGGA
GCATATCG
Cloning of p380 185 zfprrx1a_f_SalI ATTGTCGACAAGTCTCGCGCTGGCA
CCAGGCAGC
Cloning of p381 186 zfprrx1a_r_BamHI ATCGGATCCATCGAGCTCCGCATGT
GTCGCAATG
Cloning of p381 187 zfprrx1b1_f_SalI ACTGTCGACTGAGAGAAGTCGCTTG
TAGAGAAGC
Cloning of p382 188 zfprrx1b1_r_BamH ATCGGATCCACTTGCTGAGTTATAAG
CGGTGGC
Cloning of p382 189 pel2,5kb_fw_SalI GAGGTCGACGGTACAAGGCCTATCG Cloning of p383 190 pel2,5kb_rv_AgeI CAGACCGGTTCTAGAACTAGTGGAT
CC
Cloning of p383 191 SalI sHLEB 4x AAAGTCGACTCACTATAGGGCGAAT
TG
Cloning of
pTol2_4xHLEB:mClover 192 AgeI sHLEB 4x AACCGGTGATGTTCCTGGAGCTCGG
TAC
Cloning of
pTol2_4xHLEB:mClover 193 pTol_Seq1 CCTCACTTTGAGCTCCTCCACACG Sequencing
194 pTol_Seq2 GCTGGGCGCGCTCTTTTATATG Sequencing
195 pTol_Seq3 CTCAAGTAAGATTCTAGCCAG Sequencing
196 pTol_Seq4 TCGAGCCGGGCCCAAGTG Sequencing
197 pel2,5kb Seq1 CCTTTATTTTCACCTTTTCACCCCTC Sequencing of Pel2.5kb 198 pel2,5kb Seq2 GAGCTTCCACGGATTGTTGTGG Sequencing of Pel2.5kb 199 pel2,5kb Seq3 GCCGCTAATGCTACCTGTTAGCGG Sequencing of Pel2.5kb 200 pel2,5kb Seq4 GGGGCAGAGCAGCTTATCTCGGC Sequencing of Pel2.5kb 201 pTol-ERT2-Gal4 f CGAATTCGCCGCCACATGGCCGGTG
ACATGAGAG
Cloning of p396 202 pTol-ERT2-Gal4 r GTAATCCGCGGTGGCAACTTGTTTAT Cloning of p396
203 pTol-ERT2Gal4 r2 GTATCTTATCATGTCTGGATCTACTT 209 crys-RFP fw NotI AAAGCGGCCGCTCTAGATGGCCAGA
TCG
Cloning of p396 210 crys-RFP rv NotI TTTGCGGCCGCGAATTGCGCTGATG
CCCAG
Cloning of p396 211 zfprrx1a fwd AACTGAAGCGGGCAGCTGGG Prrx1a WISH probe 212 zfprrx1a rev T7 ACGTAATACGACTCACTATAGGTTCA
TCACTTACATACATGGCC
Prrx1a WISH probe 213 zfprrx1b fwd AACTAAGCCTGAAAAGAGGGAC Prrx1b WISH probe 214 zfprrx1b rev T7 ACGTAATACGACTCACTATAGGCAG
ATACATATGTCACCATGGG
239 T3 Primer AATTAACCCTCACTAAAGGG Sequencing
240 cyp26a1 fwd EcoRV ACGGAGATATCATGGGGCTGTACAC CCTTAT
Cloning of p398 241 cyp26a1 rev NotI AAAGCGGCCGCGCTAATTTCTGACA
TAACTAGTG
Cloning of p398 242 Gibson eGFP fwd TGTCAGAAATGGCGGTGGAATGGTG
AGCAAGGGCGAG
Cloning of p397 243 Gibson eGFP rev GTTCTAGAGGCTCGAAGTTTCTACTT
GTACAGCTCGTCCATGC 245 Gibson cyp26a1 rev CACCATTCCACCGCCATTTCTGACAT
AACTAGTGAATTTGGTAG
Cloning of p397
246 UAS Seq1 CCGAGCGGAGACTCTAGAGG Sequencing
247 cyp26a1 Seq1 ACCTTCTCCTGAACCTCCTC Sequencing 248 eGFP rev NotI AAAGCGGCCGCCTACTTGTACAGCT
CGTCC
Cloning of p399
249 Seq3 dnRAR GGGGAAGGTCTCTTGGTGGG Sequencing
250 Seq4 dnRAR CCCACCAAGAGACCTTCCCC Sequencing
253 4xnrUAS_dnRAR fw CTCGAAGACGCGTGGATCCAATGTA 258 ERT2-Gal4 Seq1 GCATGAAGTGCAAGAACGTGG Sequencing 259 ERT2-Gal4 Seq2 CCACGTTCTTGCACTTCATGC Sequencing 260 crys-RFP fw BglII CCCAGATCTAGATGGCCAGATCGAT
C Cloning of p440
261 crys-RFP rv BglII CCCAGATCTGAATTGCGCTGATGCCC
A Cloning of p440
262 ERT2-Gal4 Seq3 CAGGATCTCTAGCCAGGCAC Sequencing 263
ERT2-Gal4-Globin-NotI AAAGCGGCCGCCGTTGCCCAGGAGC
TGTAGG Cloning of p441
264 Gal4 DBD fw ATGAAGCTACTGTCTTCTATCGAAC Gal4 DBD WISH probe 265 Gal4 DBD rev T7 ACGTAATACGACTCACTATAGGGCG
ATACAGTCAACTGTCTTTG Gal4 DBD WISH probe 266 4xnrUAS_cyp26a1_ 270 eGFP fw SpeI CCCACTAGTATGGTGAGCAAGGGCG
AGGA Cloning of p434
271 eGFP rev SacII AAACCGCGGCTATAGGGCTGCAGAA
TCTAGAGG Cloning of p434
280 Seq_ccdB CGGGGAAGAAGTGGCTGATC Sequencing
281 Pitx1 Target 5 TAATACGACTCACTATAGGTGTGGA CTAACCTCACCGGTTTTAGAGCTAGA AATAGC
sgRNA synthesis with T7 polymerase
282 Pitx1 Target 19 TAATACGACTCACTATAGGCTGGCG CTTCTCGGCAGAGTTTTAGAGCTAGA AATAGC
sgRNA synthesis with T7 polymerase
283 Pitx1 Target 50 TAATACGACTCACTATAGGGTGGAA TGAATGTGGACAGTTTTAGAGCTAG AAATAGC
sgRNA synthesis with T7 polymerase
284 universal
bottom-strand GTTTTAGAGCTAGAAATAGCAAGTT
AAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGC TTTT
sgRNA synthesis
285 Tyr Target 1 TAATACGACTCACTATAGGACTGGA GGACTTCTGGGGGTTTTAGAGCTAG AAATAGC
sgRNA synthesis with T7 polymerase
286 Tyr T1 fw M13 TGTAAAACGACGGCCAGTAGCTCTT CAGCTCGT CTCTC
Genotyping of Tyr KO 287 Tyr T1 rv PIG GTGTCTTTGAGTGAGGATACTGC GG Genotyping of Tyr KO 288 Pitx1 T5 fw M13 TGTAAAACGACGGCCAGTCCCGAAG
AAGAAGAAGCAGCGGCGG Genotyping of Pitx1 KO 289 Pitx1 T5 rv PIG GTGTCTTCGTGTACTCTGCAGAGCTC
CTCCTG Genotyping of Pitx1 KO
290 Pitx1 T19 fw M13 TGTAAAACGACGGCCAGTGTCATTA
GATGTGGCAAAGCAGACA Genotyping of Pitx1 KO 291 Pitx1 T19 rv PIG GTGTCTTGTGCGCTTGCTTGCATGTT
C Genotyping of Pitx1 KO
292 Pitx1 T50 fw M13 TGTAAAACGACGGCCAGTCGGCTGC
TTCACGCGGTTATTC Genotyping of Pitx1 KO
293 Pitx1 T50 rv PIG GTGTCTTACACGTGAATGCGTGTATT
CCAGTGC Genotyping of Pitx1 KO
294 Seq_cmlc2 GGGGACGAACAGAAACACTGCAGA
CC
Sequencing 295 ERT2-Gal4 Seq4 GGGAGAGGAGTTTGTGTGCC Sequencing 296 Tyr T1 fw M13 (2) TGTAAAACGACGGCCAGTTGCGTAA
AGGGGGCTCTCCAGC Genotyping of Tyr KO
297 Tyr T1 rv (2) GAGCAGAGCGTCCCGGGACAC Genotyping of Tyr KO 298 Pitx1 T5 fw M13 (2) TGTAAAACGACGGCCAGTCCAAAAA
TAGCGAGTAACAGCA
Genotyping of Pitx1 KO 299 Pitx1 T5 rv (2) TTAATTTCATGCCGCAGAACAAG Genotyping of Pitx1 KO 300 Pitx1 T19 T50 fw
M13 (2) TGTAAAACGACGGCCAGTGGGTGG
GTCTCTGCGGCTCGC Genotyping of Pitx1 KO
301 Pitx1 T19 T50 rv (2) GCGAACACAGCGCAAAAGCTGGC Genotyping of Pitx1 KO 302 cyp26a1 Seq2 CCAGGTTTAACTACATCCCC Sequencing
303 4xnrUAS Seq GGTGGCTTCTAATCCGTGAGTC Sequencing 304 Seq5 dnRAR GAGACGGGACTGCTCAGTGCTATAT
GTC Sequencing
305 Seq ß-globin AAGAACAATCAAGGGTCCCC Sequencing 306 KalTA4 fw + SacI TTTGAGCTCCTCCACACGATGTACAA
GCTACTTGTTC PCR KalTA4, Restriction cloning
307 KalTA4 rv + XbaI CCATCTAGAAACTTGTTTATTGCAGC PCR KalTA4, Restriction cloning 308 IRES-mRFP rv AGGAGGCCATGGTTGTGGCCATATT
ATCATCG Cloning of p410, p411
309 IRES-mRFP fw GGCCACAACCATGGCCTCCTCCGAG G
Cloning of p410, p411 310 cyp26a1-IRES rv GGGAGAGGGGATTTCTGACATAACT
AGTGAATTTGGTAGGGAG
Cloning of p411 311 cyp26a1-IRES fw TGTCAGAAATCCCCTCTCCCTCCCCC
CC
Cloning of p411 312 cyp26a1-IRES fw 2 TGTCAGAAATTGACCCCTCTCCCTCC
CCCCC Cloning of p411
313 cyp26a1-IRES rv 2 GGGAGAGGGGTCAATTTCTGACATA
ACTAGTGAATTTGGTAGGGAG Cloning of p411 314 zfHsp70l fw_BglII CCCAGATCTGATCCTTCAGGGGTGTC
GCTTGG Cloning of 422
315 zfHsp70l_rv_SacI CCCGAGCTCCCAATAAGAGCCAAGC
CTGCAGG Cloning of 422
316 zfHsp70l fw_SalI AAAGTCGACGATCCTTCAGGGGTGT
CG Cloning of p422
317 zfHsp70l rv_SacI CCCGAGCTCCCTGCAGGAAAAAAAA
ACAATTAGAATTAATTTTATATTTA Cloning of p422 318 Seq IRES 2 GGCTCTCCTCAAGCGTATTC Sequencing 319 KalTA4 fw GCTACTTGTTCTTTTTGCAGGATCCC
ATCGATTCGAATTCGCCGCC KalTA4 WISH probe 320 KalTA4 rv T7 ACGTAATACGACTCACTATAGGGTTA
GTTACCCGGGAGGATGT KalTA4 WISH probe
321 Gibson
KalTA4-ERT2-GI fw ACTTTGAGCTCCTCCACACGGAATTC
GCCGCCACCATGA Cloning of p412-p416
322 Gibson
KalTA4-ERT2-GI rv TAAATAGATCGATCTGGCCATGGTA
CCGGGCCCAATGC Cloning of p412-p416
323 KalTA4 Seq1 CCAGTCTCTCTAGCCTGCTC Sequencing
324 Seq2 ß-globin CCAAACCGGGCCCCTCTGCT Sequencing 325 ERT2-Gal4 Seq 5 CTCGAGCCATCTGCTGGAGACA Sequencing
326 pTol_Seq5 CACCGGTTGGCTAGAGCCGGC Sequencing
327 pEX-A128 Seq1 GGCTTAACTATGCGGCATCAGAGC Sequencing 328 lac primer CACTTTATGCTTCCGGCTCGTATG Sequencing 329 ERT2-Gal4 Seq6 GTGTCTGTGATCTTGTCCAGG Sequencing 330 ERT2-Gal4 Seq7 GAGGCACACAAACTCCTCTCCC Sequencing 335 Gibson pTol-dnRAR
fw CTTTGAGCTCCTCCACACGGCGCGTG
GATCCAATGTATGAGAGTGTGG Cloning of p424 336 Gibson
ERT2-dnRAR rw ATGGCTCGAGAATCTCCATCTTCAGG
GTGATCACACGCTCCG Cloning of p424
337 Gibson
dnRAR-ERT2 fw GGAGAGGGGCTTAGAGCTCTGAGA
CTGTGGCAGGGAAAC Cloning of p424
338 Gibson IRES-ERT2
rw GGAGAGGGGCTTAGAGCTCTGAGA
CTGTGGCAGGGAAAC Cloning of p424
339 Gibson ERT2-IRES
fw AGAGCTCTAAGCCCCTCTCCCTCCCC
CCC Cloning of p424
340 Gibson
pTol-mCherry rw GTATCTTATCATGTCTGGATCTACGC
GGCCGCTTACTTGTACAGCTCG Cloning of p424 341 Gibson2 pTol-ERT2 TTGAGCTCCTCCACACGGATGCTCGA Cloning of p425
342 Gibson2
GCCGCAAACTTGTTTATTGCAGCT Cloning of p424, p425 346 Pitx1 T19 T50 fw
M13 (3) TGTAAAACGACGGCCAGGACTGACA
CGGGTTGGAGAGGA Genotyping of Pitx1 KO
347 Pitx1 T19 T50 rv (3) TAGCGAACACAGCGCAAAAGCTG Genotyping of Pitx1 KO 348 dnRAR fw SpeI CCCACTAGTATGTATGAGAGTGTGG
ATGTGAACCC Cloning of p442
349 ERT2 rw NotI AAAGCGGCCGCTTAGAGCTCTGAGA
CTGTGGCAG Cloning of p442
350 ERT2 fw SpeI CCCACTAGTATGCTCGAGCCATCTGC
TGGAGAC Cloning of p443
351 dnRAR rw NotI ACAGCGGCCGCTCAAATCTCCATCTT
CAGGGTGATCACACGC Cloning of p443
352 pTol_Seq6 TAGATCTAATCTCGACGCGCG Sequencing
353 Pitx1 T19 T50 fw
(4) GGACTGACACGGGTTGGAGAGGA Genotyping of Pitx1 KO
354 Pitx1 T19 T50 rv (3) TAGCGAACACAGCGCAAAAGCTG Genotyping of Pitx1 KO 355 ubi fw SalI CATGTCGACGCCCTTAAAACTCGAG
ACCAGC
Cloning of p423 356 ubi rw XcmI CCAACCACTATCTGGCGCCCTTTTTG
GATCCCTGTAAAC
Cloning of p423 357 Gibson ubi fw AACAGGGTAATGGGCCCCCCCTCGA
GACCAGCAAAGTTCTAGAATTTG Cloning of p423 358 Gibson ubi rw CATGTCTCCAGCAGATGGCTCGAGC
ATGTCGAATTCGCCCTTTTTGGATC Cloning of p423 359 GAVPO fw XhoI ACGTCTCGAGGATCCGCTAGCGCTA
TGAAGC Cloning of p428
360 GAVPO rw NotI AAAGCGGCCGCCCTCTACAAATGTG
GTATGGCTG Cloning of p428
361 GAVPO fw XhoI 2 CCGGCTCGAGATGAAGCTACTGTCTT
CTATCG Cloning of p428
368 IRES-mRFP.FOR GAGCTCTAAGAGCCCCTCTCCCTCCC Cloning of p423
369 IRES-mRFP.REV CTGAAAAGCCTCTCACAGGAGCGGC CGCAAACTTGT
Cloning of p423 370 Gibson ubi prom fw CGACGGTATCGATAAGCTTGATCGC
CCTTAAAACTCGAGACCAGC
Cloning of p426 371 Gibson ubi prom rv CTAGCGGATCCGCCCTTTTTGGATCC
CTGTAAAC
Cloning of p426 372 Gibson GAVPO fw AAAAAGGGCGGATCCGCTAGCGCTA
TGAAGC Cloning of p426
373 Gibson GAVPO rv GGCCATCTAGAGCGGCCGCACGCCT
TAAGATACATTGATGAGTTTGG Cloning of p426 374 Pitx1_T19_Calc_Lo
ng_1_for GGGGTTTAGATGGAAAGCTCTGCTT
G Genotyping of Pitx1 KO
375 Pitx1_T19_Calc_Lo
ng_1_rev GCCTAGCTCATTGTTTACAGCACGC Genotyping of Pitx1 KO 376 Gibson GAVPO fw ACTTTGAGCTCCTCCACACGAATTCA
TGAAGCTACTGTCTTCTATCGAAC Cloning of p417-p421 377 Gibson GAVPO rv TAAATAGATCGATCTGGCCATCTAGA
TACGCCTTAAGATACATTGATGAG Cloning of p417-p421 378 GAVPO Sonde fw ATGAAGCTACTGTCTTCTATCGAACA
AGCATGCG GAVPO WISH probe
379 GAVPO Sonde rv T7 ACGTAATACGACTCACTATAGGGTTC
CGTTTCGCACTGGAAACCC GAVPO WISH probe 382 ccdB Gibson fw AACAGGGTAATGGGCCCCCCCTCGA
GGGATCCGTCGAGATTTTCAGGAGC Cloning of p444 383 ccdB Gibson rv CTAGCCAACCGGTGGATCCACTCGA
GCCGCACCGGTCATTTATATTCCC Cloning of p444 390 Primer SP6 seq CGCCAAGCTATTTAGGTGACACTATA
G Sequencing