Construction of mRNA synthesis vectors
Yasunori Sasakura 15th December 2017
Modified 26th January 2023
There are two major vectors for in vitro mRNA synthesis for Ciona: pBS-RN3 andpBS-HTB. Both plasmids have a 5’UTR and a 3’UTR of a gene and a multicloningsite flanked by the UTRs (Figure 1). ORFs can be subcloned into the multicloning
sites of the vectors. In the mRNAs synthesized from the constructs, the UTRs
sandwich the ORF. The function of the UTRs is suspected to be the enhancement oftranslation by increasing the translation efficiency and stability of mRNAs.
pBS-RN3 was originally created by Dr. Patrick Lemaire for the synthesis of RNAsin Xenopus laevis (Lemaire et al., 1995, Cell 81, 85-94). The vector therefore uses
the UTRs of Xenopus globin. pBS-HTB was created by Dr. Hiroki Nishida for use
in Halocynthia roretzi (Akanuma et al., Dev Genes Evol 212, 459-472). The vector
uses the UTRs of Halocynthia beta tubulin.
In our experience, mRNAs created from pBS-HTB have better translation efficiency
than pBS-RN3 in Ciona intestinalis Type A/Ciona robusta. Because the difference issignificant, we recommend using pBS-HTB for this ascidian (Sasakura et al., 2010,
Mol Genet Genomics 283, 99-110).
Dr. Nishida mentioned that translation efficiency seems to be the same between
pBS-RN3 and pBS-HTB in Halocynthia. This suggests that you must test theappropriate vector for use in another ascidian. We can provide pRN3-eGFP, pHTBnEGFP, and pHTB-nKaede for the test.
The following explanation is based on pBS-HTB. You can subclone your ORF in theMCS of this vector (Figure 1) by an In-fusion reaction. The MCS includes BamHI,EcoRI, PstI, SmaI, and EcoRV restriction sites. To synthesize mRNAs, you must
linearize the vector with XhoI or KpnI/Asp3I. If your ORF has an XhoI or KpnI site,
you can subclone the ORF in the MCS of pBS-HTB-N. The vector has a NotI site at
the 3’ end of the 3’UTR, and you can linearize the vector with NotI. NotI is an 8base cutter and the recognition site is gcggccgc, which is rarely present in the Ciona
genome since the Ciona genome is rich in A and T.

mRNAs can be synthesized with T3 RNA polymerase of the mMessage mMachinetranscription kit or with the Megascript T3 kit (Ambion). When using the latter, you
may have to use a cap structure analog (S1404L or S1404S from New EnglandBiolabs), as none is supplied in the kit. The use of a poly A tailing kit is optional, but
it may enhance translational efficiency (although we did not test its effectiveness).
The following protocol is for the Megascript T3 kit.
Linearize 1-2 ug of template DNA with XhoI, Asp3I, or NotI.
Check linearization by electrophoresis.
Extract the DNA twice with phenol−chloroform.
Extract the DNA with chloroform once.
Ethanol precipitation.
Dissolve the linearized template DNA in an appropriate amount of RNase-freewater.
Reaction buffer
A solution
U solution
C solution
G solution
Cap structure analog
1 ug Template DNA + water

2 ul
2 ul
2 ul
2 ul
0.4 ul
1.6 ul
8 ul

T3 RNA polymerase
Total

2 ul
20 ul

Incubate for 5 hr at 37 oC.
Add 1 ul of DNase I (supplied in the kit).
Incubate for 15 min at 37 oC.
Add the following solutions of Poly A tailing kit (Ambion):Water
5x reaction buffer
MnCl2ATP36 ul
20 ul
10 ul
10 ul

Store 0.5 ul (RNA-A) for later comparison of the length.
Add 4 ul E-PAP, then incubate for 1 hr at 37 oC.
Take 0.5 ul of reacted solution (RNA-B) for electrophoresis. The success of the poly
A addition will be seen by the longer RNA in RNA-B relative to that in RNA-A.
Add 50 ul of LiCl precipitation solution supplied in the Megascript kit.
Centrifuge.
Wash three times with 75% ethanol to remove Li+.
Dry (Caution: never dry for too long).
Dissolve RNA in 10-20 ul water.
Vortex and put on ice.
Repeat this step several times to dissolve the RNA completely.
Measure OD 260.
Divide into aliquots and store in a deep freezer until use.

Figure 1 pBS-HTB and pBS-HTB-N
Multicloning site (MCS)
T3 promoter
5’ UTR

3’ UTR

Ampicillin-resistant gene

MCS of pBS-HTB and pBS-HTB-N:PstI
GGATCCCCGGGCTGCAGGAATTCGATATC
BamHI

SmaI

EcoRI

EcoRV

XhoI
KpnI
(pBS-HTB-N has a NotI site here)