For the construction of pTRIP-shCarabin, a DNA fragment containing H1 promoter and Carabin shRNA sequence was generated by double digestion of pSUPER-Carabin plasmid (made by inserting a shRNA targeting murine Carabin cDNA sequence (5′-GAACTACAGGATGATTCTA-3′) into pSUPER plasmid) and was subcloned within the 3′ long terminal repeat of pTRIP.CMV.GFP vector.
Because a substantial amount of positrons (material positrons) are produced when runaway electrons impact plasma-facing materials, we proposed maximizing the ratio of plasma to material positrons by inserting a thin carbon target at the plasma edge as a plasma positron bombing target and producing a plasma disruption scenario triggered by massive gas injection.
To determine whether the ratio of utilization of two AUG codons is altered by binding of an antisense LNA oligonucleotide to various locations in a 5' leader other than that of AUG1, we generated a series of mRNA constructs with isosequential 5' leaders by inserting an LNA-target sequence into different parts of the 5' leader of the (CAA)16 CAT-FLAG mRNA (Figure 5A).
To optimize the potency of different epigenetic suppressors, we constructed a target vector by inserting a GAL4-binding site cluster sequence (GBS) [ 19] upstream of a cytomegalovirus (CMV) promoter that drives the expression of the reporter luciferase gene.
We generated a new VAChT targeted mouse line by inserting a lox-P flanked TK-Neo cassette in the 3' region of the VAChT gene, in the intron between exons N and M of the ChAT gene, and a third lox-P sequence 260 bp upstream from the VAChT translational initiation codon (Figure 1).
If the recipient vector is cut between the two inverted Flp targets, Flp "heals" the double-strand break by inserting a linear fragment flanked by Flp targets.
Briefly, GDDA modifies the original target sequence by inserting a proportion of the domain sequence (10 50%) ("seed") at every amino acid position of the target sequence.
Plasmids targeting specific genes were constructed by inserting a 1 2 kb genomic PCR fragment (using primers pairs from Wormbase [ 55] of the targeted gene sequences, see Additional file 3) into plasmid L4440.
The problem of reducing the ratio of gamma to neutron yields by inserting a lead sheet after the beryllium target is studied as well.
The targeting vector was constructed by inserting a DNA fragment coding the ChR2 (a generous gift from Dr. G. Nagel, Universität Würzberg, Würzberg, Germany) fused to the Venus gene (a generous gift from Dr. A. Miyawaki, RIKEN BSI, Wako-shi, Japan) into the XhoI site of the Thy-1.2 vector (Thy-1.2-ChR2V; Fig. 1A).
