An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 247

lacZ gene was used as a reporter for gene regulation. Retroviral transduction at
low multiplicity of infection ensures integration of a single copy of the TRE-lacZ
unit into the genome. pRTonZ contains a modified neomycin phosphotransferase
gene, loxneo, in which initiating AUG has been placed upstream of a loxP site in
frame with the neo coding region. Once optimal locus is selected, it is utilized as
a target site for transgene-integration by the scheme shown in Figures 2B and 2C.
The TRE-lacZ unit is removed by Cre-mediated recombination of flanking loxP
sites, leaving one loxP site and the neo gene in the genome (Figure 2B). Since the
promoter of the loxneo gene as well as the initiating AUG are also removed, ES
cells become G418-sensitive. In this configuration, any gene of interest can be
introduced into the same locus by Cre/loxP recombination (Figure 2C). Recom-
binant ES clones can be selected by G418-resistance because the neo expression
unit is reconstituted. PCR screening showed that >90% of these G418-resistant
clones had correct insertion of the new gene.
Figure 2. Retroviral vector used to search for tightly regulated loci and strategy to introduce a new gene into
these predetermined loci. (A) Structure of the pRTonZ retroviral vector. To prevent effects of viral enhancer
on the TRE promoter, the enhancer sequence was deleted in the 3 ′ long terminal repeat (LTR). Subsequent
transduction into target cells is expected to lead to enhancer deletion in both LTRs. The insert was cloned in
the opposite orientation of the LTRs, so that the polyA addition signals would not decrease the viral titer. (B)
Excision of the lacZ reporter gene from TIGRE loci. LoxP-flanked sequences within the retroviral vector are
removed by transient expression of Cre recombinase in ES cells, leaving a single copy of loxP site in the genome.
ES cells become G418-sensitive since the loxneo gene loses its promoter and initiating AUG. (C) Introduction
of a new gene into the TIGRE locus. The G418-sensitive ES cells selected in (B) are cotransfected with the
TIGRE-targeting vector carrying a new gene (gene X) and the Cre expression vector. Proper Cre-mediated
recombination between the TIGRE-targeting vector, containing the new gene, and the TIGRE site introduces
the new gene into the TIGRE locus and converts the G418 sensitive ES cells into G418 resistant (the expected
recombinant leads to neo expression by placing the PGK promoter and initiating AUG upstream of the loxneo
gene). Symbols are: pAgl, rabbit β -globin gene polyA addition signal; P, mouse phosphoglycerate kinase-1 gene
promoter; AUG, initiating AUG of neomycin phosphotransferase gene; loxneo, neomycin phosphotransferase
gene having loxP sequence in-frame to initiating AUG; pABGH, bovine growth hormone gene polyA addition
signal; G418r, G418 resistant; G418s, G418 sensitive.