Resultados del Diagnóstico 28

The complete list of constructs used in this thesis can be found in Appendix Table A1.1. The following constructs were generated by myself, unless otherwise stated:

2.1.1 Expression Constructs

pENTR3C-Foxa2 – The 1380 bp murine Foxa2 coding DNA sequence (CDS) was amplified from pBSK-Foxa2 (Sasaki and Hogan, 1993) using oligonucleotides containing XhoI and SalI restriction sites (RA1825 and RA1824; Appendix Table A1.2). Following ammonium acetate clean up (Section 2.2.10), the Foxa2 fragment and Gateway Entry vector pENTR3C were digested with

XhoI and SalI, releasing the ccdB gene fragment from pENTR3C. The Foxa2 fragment was ligated into pENTR3C via traditional T4 ligation methods. In the subsequent pENTR3C-Foxa2 construct,

attL sites flank the Foxa2 CDS for Gateway LR based cloning.

pV5-Foxa2 – pV5-Foxa2 was generated via Gateway LR cloning (Section 2.2.14) of the pcDNA3.1- nV5-DEST (Invitrogen) Destination vector and pENTR3C-Foxa2. The Foxa2 fragment was inserted into pcDNA3.1-nV5-DEST 3’ to the V5 epitope tag.

pENTR1A-Foxj1 – The 1266 bp murine Foxj1 CDS was amplified from pYX-ASC-Foxj1 (Cruz et al., 2010) using oligonucleotides containing KpnI and NotI restriction sites (RA1545 and RA1546; Appendix Table A1.2). Following ammonium acetate clean up, the Foxj1 fragment and Gateway Entry vector pENTR1A were digested with KpnI and NotI, releasing the ccdB fragment from pENTR1A. The Foxj1 fragment was ligated into pENTR1A via traditional T4 ligation methods. In the subsequent pENTR1A-Foxj1 construct, attL sites flank the Foxj1 CDS for Gateway LR based cloning.

pV5-Foxj1 – pV5-Foxj1 was generated via Gateway LR cloning of the pcDNA3.1-nV5-DEST (Invitrogen) Destination vector and pENTR1A-Foxj1. The Foxj1 fragment was inserted into pcDNA3.1-nV5-DEST 3’ to the V5 epitope tag.

pENTR2B-NOGGIN – The 699 bp human NOGGIN CDS (Accn. No. NM_005450) was amplified from HEK293T genomic DNA via oligonucleotides containing KpnI and NotI restriction sites (RA1776 and RA1777; Appendix Table A1.2). Following ammonium acetate clean up, the

NOGGIN fragment and Gateway Entry vector pENTR2B were digested with KpnI and NotI,

62 via traditional T4 ligation methods. In the subsequent pENTR2B-NOGGIN construct, attL sites flank the NOGGIN CDS for Gateway LR based cloning.

pV5-NOGGIN – pV5-NOGGIN was generated via Gateway LR cloning of the pcDNA3.1-nV5-DEST (Invitrogen) Destination vector and pENTR2B-NOGGIN. The NOGGIN fragment was inserted into pcDNA3.1-nV5-DEST 3’ to the V5 epitope tag.

pENTR3C-ZIC2 – The 1599 bp human ZIC2 CDS was cloned into pENTR3C by Jerry Ahmed (Arkell laboratory). In the subsequent pENTR3C-ZIC2 construct, attL sites flank the ZIC2 CDS for Gateway LR based cloning.

pV5-ZIC2 – pV5-ZIC2 was generated by Radiya Ali (Arkell laboratory) via Gateway LR cloning of the pcDNA3.1-nV5-DEST (Invitrogen) Destination vector and pENTR3C-ZIC2. The ZIC2 fragment was inserted into pcDNA3.1-nV5-DEST 3’ to the V5 epitope tag.

pGEM-3’UTR-WT – the 1086 bp wildtype ZIC2 3’UTR was amplified from HEK293T genomic DNA via oligonucleotides containing XhoI restriction sites (RA1596 and RA1597; Appendix Table A1.2) and TA cloned into pGEM-T-Easy using the pGEM-T-Easy Vector System (Cat. No. A1360, Promega).

pV5-ZIC2-3’UTR – pGEM-3’UTR-WT and pV5-ZIC2 were digested with XhoI to release the ZIC2

3’UTR fragment and linearize pV5-ZIC2. The ZIC2 3’UTR fragment was gel extracted and purified, whilst pV5-ZIC2 was treated with Antarctic Phosphatase to remove the 5’ phosphate and minimise self-ligation, and purified via ammonium acetate clean up. The products were ligated via traditional T4 methods and the final pV5-ZIC2-3’UTR construct isolated via bacterial colony polymerase chain reaction (PCR). This construct was created with the help of Kathryn Dickson (Arkell laboratory).

2.1.2 Luciferase Assay Constructs

pGL4.20-DEST-luc – pGL4.20-luc was converted into a Destination vector via the Gateway® Vector Conversion System (Invitrogen). pGL4.20-luc was digested with EcoRV to linearize the vector and purified via ammonium acetate clean up. It was then taken through the Gateway® Vector Conversion System to ligate the Destination Reading Cassette A into the pGL4.20-luc

multiple cloning site (MCS).

pGL-b-globin-DEST-luc - pGL4.20-DEST-luc was digested with HindIII and BglII to linearize the vector and purified via ammonium acetate clean up. The b-globin minimal promoter was PCR amplified from pKS:b-globin:lacZ with oligonucleotides containing HindIII and BglII restriction

63 sites (RA1506 and RA1507; Appendix Table A1.2). The amplicon was subsequently digested and ligated into the linearised pGL4.20-DEST-luc via traditional ligation methods.

pGEM-T-Easy-Hsp68- The heat shock protein 68 (Hsp68) minimal promoter was PCR amplified from pBS-Hsp68 with oligonucleotides containing HindIII and BglII restriction sites (RA1640 and RA1626; Appendix Table A1.2). The amplicon was TA cloned into pGEM-T-Easy to create pGEM- T-Easy-Hsp68.

pGL-Hsp68-DEST-luc- pGL4.20-DEST-luc and pGEM-T-Easy-Hsp68 were digested with HindIII and

BglII to linearize the vector and release the Hsp68 minimal promoter, respectively. The Hsp68 minimal promoter was gel extracted and purified, and pGL4.20-DEST-luc purified via ammonium acetate clean up. The Hsp68 fragment was ligated into the linearised pGL4.20-DEST-luc via traditional ligation methods.

pGL-pZIC3-DEST-luc- pGL4.20-DEST-luc and pGl-pZIC3-luc were digested with HindIII and KpnI to linearize the vector and release the ZIC3 promoter, respectively. The ZIC3 promoter was gel extracted and purified, and pGL4.20-DEST-luc purified via ammonium acetate clean up. The ZIC3

fragment was ligated into the linearised pGL4.20-DEST-luc via traditional ligation methods.

pGL4.20-b-globin-NCE (wildtype) – pGL4.20-b-globin-NCE with the wildtype NCE was generated via Gateway LR cloning of the pGL-b-globin-DEST-luc Destination vector and pCR8-GW-TOPO- NCE. The ZIC2 NCE fragment was inserted into pGL-b-globin-DEST-luc 5’ to the promoter and

luciferase gene.

pGL4.20-Hsp68-NCE (wildtype) – pGL4.20-Hsp68-NCE with the wildtype NCE was generated via Gateway LR cloning of the pGL-Hsp68-DEST-luc Destination vector and pCR8-GW-TOPO-NCE.

The ZIC2 NCE fragment was inserted into pGL-Hsp68-DEST-luc 5’ to the promoter and luciferase

gene.

pGL4.20-pZIC3-NCE (wildtype and mutant) – pGL4.20-pZIC3-NCE with the wildtype NCE was generated via Gateway LR cloning of the pGL-pZIC3-DEST-luc Destination vector and pCR8-GW- TOPO-NCE. The ZIC2 NCE fragment was inserted into pGL-pZIC3-DEST-luc 5’ to the promoter and

luciferase gene.

2.1.3 Whole Mount In Situ Hybridization (WMISH) Riboprobes

pGEM-T-Easy-Dand5 - To make the Dand5 (previously Cerl2) probe, an 888 bp fragment of exon 2 and 3’UTR from mouse Dand5 was amplified from mouse genomic DNA (NCBI Accn. No: NR033145) using oligonucleotides RA1570 and RA1571 (Appendix Table A1.2) and TA cloned into pGEM-T-Easy using the pGEM-T-Easy Vector System (Cat. No. A1360, Promega).

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pGEM-T-Easy-Rfx3 - To make the Rfx3 probe, a 472 bp fragment of exon 16 and the 3’UTR of mouse Rfx3 was amplified from mouse genomic DNA (NCBI Accn. No. NM011265) using oligonucleotides RA1592 and RA1593 (Appendix Table A1.2) and TA cloned into pGEM-T-Easy using the pGEM-T-Easy Vector System.

pGEM-T-Easy-Noto - To make the Noto probe, a 386 bp fragment of the 5’UTR and exon 1 of mouse Noto was amplified from mouse genomic DNA (NCBI Accn. No. NM001007472.2) using oligonucleotides RA1590 and RA1591 (Appendix Table A1.2) and TA cloned into pGEM-T-Easy using the pGEM-T-Easy Vector System.

2.1.4 PiggyBac Transposon Constructs

pBB262-lacZ - p1229 and pBB262 were digested with XbaI and XhoI. The lacZ fragment released from p1229 was gel extracted (Section 2.1.9) and ligated into linearized pBB262. Following electrotransformation into DH5aEscherichia coli (E. coli), blue-white colony screening was used to select colonies containing the pBB262-lacZ construct.

pBB262-ZIC2 NCE (wildtype and mutant) - The 608 bp wildtype ZIC2 NCE, and each of the six mutated versions of the ZIC2 NCE, were PCR amplified from the pCR8-GW-TOPO-NCE constructs as described in Section 2.2.13, using oligonucleotides containing XbaI restriction sites and homology to the pBB262 MCS at the 5’ ends (RA1443 and RA1464; Appendix Table A1.2). An artificial NotI site was also included in oligonucleotide RA1464 to facilitate future lacZ cloning. pBB262 was digested with Xbal in the MCS to linearize the plasmid, and the NCE amplicons were cloned into pBB262 via In-Fusion Cloning (Section 2.2.12), resulting in wildtype and NCE mutant pBB262-NCE constructs (Appendix Figure A7.3).

Wildtype and mutant pBB262-NCE-lacZ - Following isolation and large scale prepping, pBB262-

ZIC2 NCE constructs were digested with NotI and XhoI to linearize the vector. p1229, a Bluescript II KS vector containing a 4354 bp lacZ reporter/SV40 PolyA cassette, was digested with NotI and

XhoI to release the lacZ/SV40 PolyA. This was subsequently ligated into the digested pBB262- NCE constructs via traditional T4 ligation as described in Section 2.2.11. Following electrotransformation into DH5a E. coli, blue-white colony screening (Section 2.2.4) was used to select colonies containing wildtype and mutant pBB262-NCE-lacZ constructs.

pBB262-mNet – pmNetg and pBB262were digested with KpnI and NotI sites to release the 1617 bp mNet fragment and linearize the vector, respectively. Following gel extraction and purification, the mNet fragment was ligated into pBB262 via traditional T4 ligation as described in Section 2.2.11.

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pBB262-mNet-lacZ – Following isolation and large scale prepping, the pBB262-mNet construct was digested with NotI and XhoI to linearize the vector. p1229, a Bluescript II KS vector containing a 4354 bp lacZ reporter/SV40 PolyA cassette, was digested with NotI and XhoI to release the lacZ/SV40 PolyA. This was subsequently ligated into the digested pBB262-mNet construct via traditional ligation. Following electrotransformation into DH5aE. coli, blue-white colony screening (Section 2.2.4) was used to select colonies containing the pBB262-mNet-lacZ

construct.