An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 253

production of iKO mice, we have utilized a large-scale insertional mutagenesis
ES cell library developed in house for the KO production [18]. Figure 6A upper
panel illustrates the structure of the retroviral vector used. In the particular case
of ApoE, the virus is inserted in the third intron. The vector contains splice ac-
ceptor, stop codons, polyA signal and transcriptional terminator to ensure gene
inactivation, which we confirmed to be the case by showing that ~90% of isolated
gene-specific ES clones were null alleles [18]. (The remaining were mostly knock-
downs, and in nearly all these cases the retrovirus was inserted into 5 ′ UTR (exon
or intron), suggesting that retroviral insertions upstream of the coding regions of
genes should be avoided.) The vector also includes the rtTA gene immediately
downstream of the splice acceptor, stop codons and internal ribosome entry site
(IRES), so that rtTA protein can be synthesized from the ApoE-IRES-rtTA hy-
brid transcript.
To examine if random insertion of the retroviral vector containing rtTA into a
gene results in rtTA expression reflecting the expression patterns of the inactivated
gene, we compared rtTA transcription with the transcription of the endogenous
gene by RT-PCR in different tissues for 26 different G protein coupled receptor
(GPCR) KO lines generated from the library. Heterozygous mice carrying one al-
lele of the intact endogenous gene and one allele interrupted by the rtTA-bearing
retroviral vector were used to prepare total RNA samples from different tissues
and amplify gene-specific transcript and rtTA transcript from the same RNA
preps for side-by-side comparison. Figure 6B shows three examples of such com-
parison for genes P2Y6, RE2 and LGR6 respectively. The retroviral vector was
inserted into a different part of each of the three genes - a 5 ′ UTR intron of P2Y6,
an intron within the coding region of RE2 and the 15th coding exon of LGR6. In
all three cases, rtTA expression profiles closely resemble those of the endogenous
genes to be inactivated. Real-time qPCR for 16 tissues also showed high degree
of correlation between rtTA and endogenous gene from tissue to tissue. Overall,
out of the 26 lines examined, 19 lines showed good correlation between rtTA and
the endogenous gene's expression , i.e. rtTA is expressed in the tissues where the
corresponding endogenous gene is expressed and the relative ratio across differ-
ent tissues for each transcript also appears similar between rtTA and endogenous
gene. In the remaining 7 lines in which rtTA was not expressed well, 5 lines had
the retroviral vector inserted upstream of the first (and usually fairly large) intron
of each gene. It has been recognized that the first intron (especially if it is large)
could contain essential transcriptional regulatory elements, and thus insertions in
this region might disrupt the transcriptional regulation and so should be avoided.
Otherwise, our data showed that in the majority of insertional sites, rtTA could be
expressed through the endogenous promoter upon integration via the retroviral
vector.