Cloning of Ci-multidom
Isolation of 204d, an incomplete cDNA clone of Ci-multidom, has been reported previously (Hotta et al.,1999). Rapid amplification of cDNA 5′-ends (5′RACE) was performed using the GeneRacer™ kit (Invitrogen, CA). For 5′RACE, total RNAs from early tailbud stage embryos were reverse-transcribed using oligo(dT)12–18 primers in combination with the following specific primers:
Ci-multiL496 5′-CAAAGCTGCAGACACTGCCTTCG-3′ and
The relative positions of these primers along the gene model are indicated in Figure 1A.
Electroporations were performed according to previously published protocols (Corbo et al.,1997; Bertrand et al.,2003), with some slight modifications. One hundred microliters of fertilized eggs were mixed with 250 μl of 0.96 M mannitol containing 50 μg of plasmid DNA, and electroporated in 4 mm cuvettes with a Gene Pulser Xcell System (BIO-RAD), using a square pulse protocol (50V and 16 ms per pulse). After electroporation, embryos were allowed to develop until the tailbud stage and fixed in filtered seawater containing 0.2% glutaraldehyde for 30 min at room temperature, then washed in PBST (phosphate-buffered saline containing 0.1% Tween20) twice and washed once in 1 mM MgCl2, 3 mM potassium ferrocyanide and 3 mM potassium ferricyanide in PBST for 5 min. The staining reactions were carried out in PBST containing 400 ng/ml 5-bromo-4-chloro-3-indolyl—D-galactopyranoside (X-gal), 1 mM MgCl2, 3 mM potassium ferrocyanide, and 3 mM potassium ferricyanide at 25°C for ∼12 hr, then stopped by washing in PBST.
To construct the Ci-Bra>EGFP plasmid, the EGFP coding sequence was PCR-amplified from the EGFP-C1 plasmid (Clontech) using the following primers:
5′-GAAGGATCCGATGGTGAGCAAGGGCGAGG-3′ (EGFP forward) and
5′-TTAGCGGCCGCTTACTTGTACAGCTCGTCCATGC-3′ then cloned into the BamHI and NotI sites of Ci-Bra>GFP(NotI), which was constructed by inserting a NotI linker into the BlpI site of the 3.5-kb Ci-Bra>GFP plasmid (Corbo et al.,1997). A BamHI/NotI fragment containing the full-length Ci-multidom ORF was ligated into the BamHI and NotI sites of the Ci-Bra>GFP(NotI) vector to make the Ci-Bra>Ci-multidom plasmid.
To generate the Ci-Bra>EGFP-multidom plasmid, the EGFP coding sequence was amplified using the EGFP forward primer and the following reverse primer:
5′-TTAGGATCCTTGTACAGCTCGTCCATGC-3′ and then cloned into the BamHI site of the Ci-Bra>Ci-multidom plasmid.
To create the Ci-Bra>Nup50-mRFP plasmid, the Ci-Nup50 coding sequence was PCR-amplified from EST clone CiGC29i23, obtained from the Ciona intestinalis Gene Collection Release 1 (Satou et al.,2002; courtesy of Dr. Nori Satoh), using the following primers:
The resulting PCR product was then inserted into the BamHI and NotI sites of the Ci-Bra>mRFP plasmid (Rhee et al.,2005).
To create the Ci-Bra>KDELR-mRFP, the mRFP coding sequence and Ci-KDELR coding sequence were inserted into the BamHI/NotI sites and SalI/BamHI sites of Ci-Bra-MCS, respectively. To make the Ci-Bra-MCS plasmid, in which a multicloning site is inserted downstream of the 3.5-kb Ci-Bra regulatory region, a PstI-BlpI-SalI-BamHI-NotI linker was inserted into the PstI and NotI sites of the Ci-Bra>GFP (NotI) vector. The mRFP coding sequence was PCR-amplified from pCX-myrmRFP1 plasmid using the following primers:
Ci-KDELR (gene model: ci0100149851) was cloned by RT-PCR using the following primers:
5′-TTGTGGATCCTGCAGGAAGGCTAAGCTTCTT-3′ and reverse transcribed using as a template mRNAs from early tailbud Ciona embryos.
To create the Ci-Bra>ST-mRFP plasmid, the membrane anchor sequence from rat sialyltransferase (Boevink et al.,1998) was amplified from the ST-aGFPM5 plasmid (Zeller et al.,2006) using the primers:
5′-ATCGGATCCCATGATTCACACCAACCTGAAG-3′ and 5′-TGAGCGGCCGCCCAAGCTTCCTTGGTTTGCAA-3′ and was inserted into the BamHI and NotI sites of the Ci-Bra>mRFP plasmid (Rhee et al.,2005). The Ci-sna>Ci-Bra plasmid was prepared by cloning the Ci-Bra coding region into the PstI and BlpI sites located at the 3′-end of a 1-kb fragment from the Ci-snail (ci0100151261) 5′-flanking region (in pSP72-1.27; Corbo et al.,1997). The Ci-sna 1-kb fragment encompasses the 504-bp muscle enhancer described by Erives et al. (1998).
To create the enhancer construct 1, which includes the putative endogenous Ci-multidom promoter, a 1.1-kb fragment upstream of Ci-multidom was PCR-amplified from Ciona genomic DNA using the following primers:
5′-ATGGGATCCAATACGGATCGGTTTCTTTTAA-3′ (-1.1 forward) and 5′-TGTGCGGCCGCCTTTGTGTTCTCGTTATTTCTCC-3′ then cloned into the BamHI and NotI sites of the pFBΔSP6 plasmid, which carries the LacZ reporter gene. The pFBΔSP6 plasmid contains 155 bp of the Ci-fkh/HNF-3β(Ci-fkh; JGI gene model ci0100153163; Di Gregorio et al.,2001) basal promoter and part of the first exon of Ci-fkh fused in frame to the lacZ reporter gene and inserted into the pSP72-1.27 vector (Harafuji et al.,2002). Since the SP6 site originally present in pSP72-1.27 was found to contain a T-box binding site, the Ci-fkh basal vector was cut with Eco0109I and XhoI, then filled in and relegated. This created a deletion of 282 bp, which removes the SP6 site without affecting the ability of the resulting vector to replicate itself. By cutting with BamHI and NotI, the Ci-fkh basal promoter was removed and replaced by the alternative promoter regions. To create the enhancer construct 1, which contains the −1.1-kb intergenic fragment upstream of the Ci-fkh basal promoter, the 1.1-kb fragment was PCR-amplified from Ciona genomic DNA using the −1.1 forward primer and the following reverse primer:
5′-GGTCCATGGTACCCGCGTAACTTTCTTGCA-3′ and then cloned into the BamHI and NcoI sites of the pFBΔSP6 plasmid.
Finally, as a first step to create enhancer construct 3, the Ci-Bra promoter region was PCR-amplified from Ciona genomic DNA using the primer:
5′-TGTCCATGGCTGTATAAACTTGCACCCGAG-3′ and the following reverse primer:
5′-AATGCGGCCGCTGATGACGTCATTATAGGTTTGT-3′ and then it was cloned into the NcoI and NotI sites of the pFBΔSP6 plasmid to replace the Ci-fkh basal promoter and to generate the pBrapro vector. Subsequently, the 1.1 intergenic fragment was PCR-amplified using the −1.1 forward primer and the following reverse primer:
The resulting product was cloned into the BamHI and NcoI sites of the pBrapro plasmid.
Total RNAs were isolated from 10 μl of mid-tailbud embryos using the RNeasy Protect Mini Kit (Qiagen), according to the manufacturer's instructions. RNA samples were resuspended in RNAse-free water and quantified spectrophotometrically at 260 nm. Five hundred ng of total RNAs were reverse transcribed in a 20-μl total volume, using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen) and the supplied oligo(dT)12–18 primer; a negative control, which was incubated in parallel but did not contain reverse transcriptase, yielded no products.
All PCR amplifications were performed in a 25-μl total volume using Taq DNA polymerase (New England Biolabs), 5× Green GoTaq Reaction Buffer (Promega) and 0.5 μl of the synthesized cDNA sample as a template. The number of cycles was optimized for each primer pair to remain within the linear range of amplification.
Control PCR reactions, performed in parallel without reverse transcriptase, did not amplify any product. Gel electrophoresis of 1 or 2 μl of each PCR reaction was carried out on 2.0% agarose gels. After electrophoresis, digital images of the gels were acquired using a Kodak EDAS 290 imaging system. The relative levels of intensity of the bands obtained were quantified using the Multi-Analyst version 1.0.2 software (Bio-Rad). The primers used for RT-PCR were as follows:
for Elongation factor 2 (EF2): 5′- CAAAAGAGGGTGTCCTTTGTG-3′ and 5′-GGAGTTCCAGGGTTATGTGC-3′; for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH):
5′-GCAGGAGCTGGCATTGCAC-3′ and 5′-ACAGTAAGATTGCATGCAACAG-3′; for Ci-multidom: 5′-TGTCTGCAGCTTTGAATGTTAC-3′ and 5′-GGGTTGTCCCATTTCATATCAC-3′.
Histochemistry and Confocal Microscopy
Embryos were fixed with 4% paraformaldehyde in 0.5 M NaCl and 0.1 M MOPS pH 7.0 at room temperature for 30 min. To visualize the cell membranes by staining the actin cytoskeleton, embryos were rinsed with PBST (phosphate-buffered saline containing 0.1% Tween20) 3 times and incubated in 1 U (200 μl) of Rhodamine-phalloidin (Invitrogen) in PBS/0.2% Triton X100 for 3 hr at room temperature. After incubation, embryos were washed 3 times with PBST and mounted on microscope slides with Vectashield Mounting Medium (Vector Laboratories, CA). To visualize the nuclei, embryos were rinsed 3 times with PBST and incubated in 5 μM TO-PRO-3 iodide (Invitrogen) in PBS/0.2% Triton X100, overnight at 4°C. After incubation, embryos were washed and mounted as described above. Embryos electroporated with Ci-Bra>ST-mRFP were fixed with 100% acetone for 30 min at −20°C.
Laser scanning confocal images were acquired with a Zeiss LSM 510 on a Zeiss Axiovert 200 widefield microscope, using either a 63× or a 25× oil immersion lens. Confocal microscopy was carried out at the Weill Cornell Medical College Optical Microscopy Core Facility.