Pcag gfp vector

To compare the efficiency of ET by SB, PB and Tol2 transposons, the GFP trapping box containing mini-promoter Krt4 (keratin4), GFP and β-globin polyA was constructed. The Krt4 minimal promoter was cloned from the zebrafish genome using primers 5′-gcACTAGTAAGCTTgtgtgtgtgtgagagcagtc-3′ and 5′-atGAATTCaggtacgagagtgctctctg-3′, and the CAG promoter of pCAG-GFP vector (Addgene: 11150) was replaced with SpeI/EcoRI sites. Then, the GFP trapping box was subcloned into the SB, PB and Tol2 transposon structural frames and named pSB-Krt4-GFP, pPB-Krt4-GFP and pTol2-Krt4-GFP, respectively (Figure 1a). The SB100X transposase plasmid was a gift from Dr. Zoltan Ivics (Paul Ehrlich Institute, Germany); the PB transposase plasmid was a gift from Dr. Allan Bradley (Wellcome Trust Sanger Institute, UK); the Tol2 transposase plasmid was received from Vladim Korzh (National University of Singapore, Singapore); the SB100X and PB and Tol2 ORFs were subcloned into the pTNT^TM vector, while pTNT^TM plasmid was ordered from (AL5610; Promega, Fitchburg, WI, USA). We named them pTNT-SB100X, pTNT-PB and pTNT-Tol2. Then, transposase mRNA was synthesized in vitro using a mMESSAGE mMACHINE T7 Kit (AM1344; Ambion, Austin, Texas, USA) from the pTNT-SB100X/PB/Tol2 constructs according to the kit instructions.

According to Chung, Bell & Felsenfeld (1997) (link), the 5′HS4 insulator, which contains two sequentially connected insulator sequences, was cloned from the chicken genome by high-fidelity PCR using the primers InsF and InsR (Table 1). Then, the 5′HS4 element was inserted upstream and downstream of the mCherry expression box in the ET vectors described above, and the resulting enhancer-detection vectors were named pEDV-Gata and pEDV-Krt4.
Four enhancers (Z48, Hand2, CNS1 from the zebrafish genome and Hs769 from the human genome) were cloned by high-fidelity PCR using primers designed according to the GenBank sequences (Table 1).The correct enhancers were sub-cloned upstream of the minimal promoter in pEDV-Gata or pEDV-Krt4 using the EcoRl and Agel restriction sites.
The mouse Beta-globin minimal promoter was cloned by extending PCR using primers designed according to Carter et al. (2002) (link). This promoter was then inserted upstream of the GFP reporter gene, to replace the CAG promoter in the pCAG-GFP vector (11150; Addgene, Watertown, MA, USA). The GFP expression box driven by the Beta-globin minimal promoter was flanked by the 5′HS4 insulators, to generate another enhancer-detection vector named pEDV-Beta-globin-GFP.

cDNAs encoding Psma8 and Psma7 were PCR amplified from mouse testis cDNA and cloned into a pCAG-GFP vector (Addgene #11150) for C-terminal green fluorescent protein (GFP) fusion. After the maxi-prep procedure (Qiagen, #12163), 50 μg of plasmids were injected into live mouse testes at PD16–18. One hour after injection, the testes were electroporated via four forward and four reverse pulses (50 with 950 ms intervals) at a voltage of 30 V. Mice were sacrificed 7 days after electroporation and subjected to preparation of western blotting samples and testes sections.