Glowing zebrafish: Integration, transmission, and expression of a single luciferase transgene promoted by noncovalent DNA–nuclear transport peptide complexes

Abstract

The development of vehicles driving foreign DNA into the cell nucleus is essential for effective cellular gene transfer applications. We report that noncovalent binding of nuclear localization signal (NLS) peptides to plasmid DNA enhances nuclear uptake of the DNA and promotes germline integration, inheritance, and expression of a single copy of a luciferase reporter gene in zebrafish. As few as 10 DNA-NLS complexes (0.06 fg plasmid DNA) cytoplasmically injected are sufficient to produce germline-transgenic zebrafish bearing a single copy of the transgene. This corresponds to a 10(5)-fold reduction in DNA concentration compared to commonly used procedures. Use of 10(3) or 10(4) DNA-NLS complexes augments the number of transgene integrations, which occur mostly within 1-4 distinct insertion sites in the genome. In situ hybridization analyses and transmission studies show that transgene integration into the germline and somatic tissues is mosaic, and that the extent of mosaicism is negatively correlated with the amount of DNA-NLS injected. In addition, a larger proportion of zebrafish harboring a single copy of the transgene expresses luciferase, albeit at a 10-fold lower level than those containing numerous transgene insertions. The data demonstrate the potential use of nuclear targeting peptides noncovalently bound to vector DNA to enhance the efficiency of biotechnological nonviral gene transfer applications.

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno‐associated virus in zebrafish

“…This differs from the results of other studies, in which transgenes often had multiple integration patterns in F1 progeny derived from the same founder (Alam et al, 1996;Culp et al, 1991;Liang et al, 2000;Penman et al, 1991). This clonal integration pattern makes it much easier to establish stable homozygotes.…”

“…Cytoplasmic microinjection of transgenes results in highly mosaic founder gonads and low germ-line transmission rates. Thus, many progeny must be screened by dot blot or PCR to find transgenic founders (Stuart et al, 1988;Culp et al, 1991;Liang et al, 2000). A reporter gene helps reduce the screening effort and makes it possible to monitor the expression pattern of the transgene noninvasively.…”

“…The BAC construct is suitable for targeting gene expression, but it is not always necessary for mapping promoter activity, because tissue-and stage-dependent cis-acting elements may be located in the proximal region of the promoter. Binding nuclear localization signal peptides to plasmid DNA also enhanced transient expression and increased the efficiency of gene transfer and germ-line transmission (Collas and Alestrom, 1998;Liang et al, 2000). Reporter gene expression in the F0 generation was still mosaic, however, and the transgene was not expressed in some transgenic progeny.…”

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

“…They suggested that the NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in several species. Several reports confirm the potential use of nuclear targeting peptides, which are non-covalently bound to vector DNA, to enhance the efficiency of biotechnological non-viral gene transfer applications (Malecki, 1996;Aronsohn and Hughes, 1998;Schwartz et al, 1999;Liang et al, 2000;Chan and Jans, 2001).…”

Cationic lipid-based delivery system for systemic cancer gene therapy

“…Second, the transfection efficiency needs to be increased and/or expression of transgenes with increased potency needs to be optimized to completely suppress tumor growth rather than simply decrease the growth rate. Examples of technology that can overcome these limitations are reduction of nonspecific cell interactions with masking technologies, such as incorporating polyethylene glycol; transfection complex targeting with ligands to yield selective uptake to tumor vasculature; coformulation of agents to facilitate endocytic vacuole escape of the delivered gene, such as pH-dependent lytic peptides 21,22 or proton-absorbing polymers; 23 and increased nuclear uptake of the plasmid either by incorporating nuclear localization agents 24 or by simply including DNA sequences that endogenous nuclear localization proteins can recognize. 25 There are several alternative types of genes that can be expressed with increased potency.…”