PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

Abstract

Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.

Deregulated expression of Cdc6 in the skin facilitates papilloma formation and affects the hair growth cycle

“…[24][25] Upon pronuclear injection of the construct into fertilized eggs and implantation into stimulated females, a K5-Cdc6 transgenic strain was established. Southern blot analysis indicated tandem integration of K5-Cdc6 copies, a common event in microinjection-mediated transgenesis 26 ( Fig. S1A).…”

<i>Thy1</i> promoter activity in the <i>Rosa26</i> locus in mice: lessons from Dre-<i>rox</i> conditional expression system

“…Thy1 promoter activities at the Rosa26 locus were examined using Tg mice generated by PITT/i-PITT. The Thy1 promoter is frequently utilized in Tg mice generated by traditional random integration transgenesis [10][11][12][13]. These Tg mice are mainly used for analyses of gene functions in the brain, where the Thy1 promoter is predominantly active.…”

“…The production of Tg mice has long relied on direct microinjection (MI) of a DNA fragment into the pronuclei of fertilized eggs. This generates random integration of the transgene (or genes) with multicopy configuration into host chromosomes, often leading to unreliable and unstable expression of the transgene [13]. Researchers must thus select appropriate Tg lines with desired transgene expression patterns that faithfully recapitulate endogenous gene expression.…”

“…Unfortunately, this selection procedure requires considerable time, effort, and cost. Embryonic stem cell-mediated transgenesis has been developed as an alternative strategy to generate Tg mouse lines, but this procedure is costly and laborious [13].…”

Insertion of sequences at the original provirus integration site of mouseROSA26 locus using the CRISPR/Cas9 system

“…2A). While there were many founders that had the expected increase in size, some (founders 6,9,14,15,16, 20 and 23) had higher than the expected sized bands ( Fig. 2A).…”