Sense RNA constructs

Construct Vector Restriction

enzyme Polymerase Reference

Ptf1a pCS2+GR NotI SP6 (Afelik et al.,

2006)

Ptf1a^W242A pCS2+GR NotI SP6 (Hanotel et al.,

2014) Ptf1aW224A/W242A pCS2+GR NotI SP6 (Hanotel et al.,

2014)

Ptf1a^T243A pCS2+GR NotI SP6 This thesis

Ptf1a^T243E pCS2+GR NotI SP6 This thesis

Ptf1a-Vn pCS2+Vn NotI SP6

(Hedderich, unpublished

data) Ptf1aW224A/W242A

-Vn pCS2+Vn NotI SP6

(Hedderich, unpublished

data)

Ptf1a^T243A-Vn pCS2+Vn NotI SP6 This thesis

Ptf1a^T243E-Vn pCS2+Vn NotI SP6 This thesis

Rbpj-Vc pCS2+Vc NotI SP6

(Hedderich, unpublished

data)

Prdm13-Vc pCS2+Vc NotI SP6 This thesis

Neurog2-Vn pCS2+Vn NotI SP6 This thesis

Table 2.4 List of overexpression constructs

Overexpression constructs:

E12-VcpCS2+ (prepared by M. Hedderich and K. Ditter):

E12-VcpCS2+ contains the open reading frame of Rbpj fused at its N-terminus with the C-terminus of Venus YFP (aa 155-238). E12-VcpCS2+, was generated by PCR amplification of the E12 open reading frame using XE12pSP64XBm (obtained from Eric Bellefroid) as a template and the following primers: for, 5´-GTACCGGTGGTGGTGGTGGTGGTAATCAGCAG CAGAGGATG-3´ and rev, 5´-GGGTACGTATCACATGTGTCCCACTGG-3´.

The PCR product was inserted into VcpCS2+ (Saka et al., 2007) using AgeI and SnaBI restriction sites. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Ptf1a-VnpCS2+ (prepared by M. Hedderich and K. Ditter):

Ptf1a-VnpCS2+ contains the open reading frame of Ptf1a fused at its N-terminus with the N-N-terminus of Venus YFP (aa 1-154). Ptf1a-VnpCS2+, was generated by PCR amplification of the Ptf1a open reading frame using Ptf1a-GRpCS2+ (Afelik et al., 2006) as a template and the following primers: for, TACCGGTGGTGGTGGTGGTGGTGAAACGGTCCTGGAGC-3´ and rev, 5´-GGGTACGTATCACATATCAAGGCACAAAG-3´. The PCR product was inserted into VnpCS2+ (Saka et al., 2007) using AgeI and SnaBI restriction sites. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

E12-Vc pCS2+Vc NotI SP6

(Hedderich, unpublished

data)

β-Gal pCS2+ NotI SP6 (Chitnis and

Kintner, 1995)

NotI SP6 (Bier et al.,

1989)

mRFP pCS2+ NotI SP6 (Saka et al.,

2007)

Rbpj-VcpCS2+ (prepared by M. Hedderich and K. Ditter):

Rbpj-VcpCS2+ contains the open reading frame of Rbpj fused at its N-terminus with the C-terminus of Venus YFP (aa 155-238). Rbpj-VcpCS2+, was generated by PCR amplification of the Rbpj open reading frame using Rbpj-GRpCS2+ (Hedderich, 2012) as a template and the following primers: for, 5´-GTACCGGTGGTGGTGGTGG TGGTCAACCTGGCATTCCTAAATAC-3´ and rev, 5´-GGGTACGTATTAGGACACTACTGCTGCAGTG-3´. The PCR product was inserted into VcpCS2+ (Saka et al., 2007) using AgeI and SnaBI restriction sites. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Ptf1aW224A/W242A-VnpCS2+ (prepared by M. Hedderich and K. Ditter):

Ptf1aW224A/W242A-VnpCS2+ contains the open reading frame of Ptf1a fused at its N-terminus with the N-terminus of Venus YFP (aa 1-154). Ptf1aW224A/W242A -VnpCS2+, was generated by PCR amplification of Ptf1a open reading frame using Ptf1aW224A/W242A-GRpCS2+ (Hedderich, 2008) as a template and the following primers: for, 5´-TACCGGTGGTGGTGGTGGTGGTGAAACGGT CCTGGAGC-3´ and rev, 5´-GGGTACGTATCACATATCAAGGCACAAAG-3´.

The PCR product was inserted into VnpCS2+ (Saka et al., 2007) using AgeI and SnaBI restriction sites. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Ptf1a^T243A-GRpCS2+ (prepared by M. Hedderich and K. Ditter):

Ptf1a^T243A-GRpCS2+ contains the open reading frame of Ptf1a with putative phosphorylation site threonine 243 mutated to alanine. To synthesize Ptf1a^T243A-GRpCS2+, the threonine residue in position 243 was mutated using the QuickChange XL Site Directed Mutagenesis Kit (Stratagene) and the following primers (mutations are indicated in red): for, 5´-GGCCAAAGTGTGG GCTCCTGAGGATCCC-3´ and rev, 5´-GGGATCCTCAGGAGCCCACAC TTTGGCC-3´ and Ptf1a-GRpCS2+ as a template. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Ptf1a^T243E-GRpCS2+:

Ptf1a^T243E-GRpCS2+ contains the open reading frame of Ptf1a with putative phosphorylation site threonine 243 mutated to glutamate. To synthesize Ptf1a^T243E-GRpCS2+, the threonine residue in position 243 was mutated using the QuickChange XL Site Directed Mutagenesis Kit (Stratagene) and the following primers (mutations are indicated in red): for, 5´-CGGC CAAAGTGTGGGAGCCTGAGGATCCCAGG-3´ and rev, 5´-CCTGGGATCC TCAGGCTCCCACACTTTGGCCG-3´ and Ptf1a-GRpCS2+ as a template. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Ptf1a^T243A-VnpCS2+:

Ptf1aT243A-VnpCS2+ contains the open reading frame of Ptf1a^T243A fused at its N-terminus with the N-terminus of Venus YFP (aa 1-154). To synthesize Ptf1a^T243A-VnpCS2+, the threonine residue in position 243 was mutated using the QuickChange XL Site Directed Mutagenesis Kit (Stratagene) and the following primers (mutations are indicated in red): for, 5´-GGCCAAAGT GTGGGCTCCTGAGGATCCC-3´ and rev, 5´-GGGATCCTCAGGAGCCC ACACTTTGGCC-3´ and Ptf1a-VnpCS2+ as a template. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Ptf1a^T243E-VnpCS2+:

Ptf1a^T243E-VnpCS2+ contains the open reading frame of Ptf1a^T243E fused at its N-terminus with the N-terminus of Venus YFP (aa 1-154). To synthesize Ptf1a^T243E-VnpCS2+, the threonine residue in position 243 was mutated using the QuickChange XL Site Directed Mutagenesis Kit (Stratagene) and the following primers (mutations are indicated in red): for, 5´-CCTGGGATC CTCAGGCTCCCACACTTTGGCCG-3´ and rev, 5´-CGGCCAAAGTGTG GGAGCCTGAGGATCCCAGG-3´ and Ptf1a-VnpCS2+ as a template. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Neurog2-VnpCS2+:

Neurog2-VnpCS2+ contains the open reading frame of Neurog2 fused at its N-terminus with the N-terminus of Venus YFP (aa 1-154). Neurog2-VnpCS2+, was generated by PCR amplification of the Neurog2 open reading frame using Neurog2-HApCS2+ (Hedderich, unpublished data) as a template and the following primers: for, 5´-GTACCGGTGGTGGTGGTGGTGGTGTGCT GCTCAAGTGCG-3´ and rev, 5´-GGGTACGTATCAAATGAAAGCGCTGCT-3´. The PCR product was inserted into VnpCS2+ (Saka et al., 2007) using the AgeI and SnaBI restriction sites. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.

Prdm13-VcpCS2+:

Prdm13-VcpCS2+ contains the open reading frame of Prdm13 fused at its N-terminus with the C-N-terminus of Venus YFP (aa 155-238). Prdm13-VcpCS2+, was generated by PCR amplification of the Prdm13 open reading frame using cDNA as a template and the following primers: for, 5´-ATGCATTGCAACAGGGCTC-3´ and rev, 5´-TTAGGGTTCCTTGCTG CTTCC-3´. The insert was subcloned into pGemTeasy vector, which was then used as a template for PCR amplification of the Prdm13 open reading frame using the following primers: for, 5´-GTACCGGTGGTGGTGGTGGTGGTC ATTGCAACAGGGCTCTG-3´ and rev, 5´-GGGTACGTATTAGGGTTCC TTGCTGCTTC-3´. The PCR product was inserted into VcpCS2+ (Saka et al., 2007) using the AgeI and SnaBI restriction sites. For preparation of sense RNA, the construct was linearized with NotI and transcribed with SP6 polymerase.