Mice with Cre-inducible Adipoq have become a popular model to study adipose tissue biology1,2. Although early data from SNP panel analysis have suggested possible integration of the BAC on chromosome 9, the exact integration site has not yet been established. As a result, Wong et. al sought to resolve the genomic position of the integration event as well as determine the integrity of the integrated transgenic sequence.3
Limitations of conventional gene sequencing methods
Challenges in using traditional sequencing methods to determine this insertion site, include the large transgene size, potential recombination, and the high degree of homology between the DNA sequence of the transgene with that of the endogenous locus at chromosome 16. In fact, designing effective conventional genotyping primers has proven to be cumbersome and unsuccessful. Furthermore, the use of a SNP panel does not allow discriminating between heterozygous and homozygous animals. Given the absence of an established technique, most studies and publications therefore involve the use of hemizygous Tg(Adipoq-Cre)1Evdr mice.
Advantages of TLA analysis for genomic screening
In this study, 5 TLA primer sets were used to ensure sufficient coverage across the entire transgene/integrated vector sequence. TLA data confirmed that the transgene sits on chromosome 9, and more precisely in the intron between exons 6 and 7 of the Tbx18 gene. Of note, this gene plays a key role in embryonic development, by ensuring proper myocardial function4,5,6. Importantly, TLA did not reveal any large structural rearrangements, accompanying recombination, in the host genome at the integration site.
Moreover, TLA detected transgene-transgene fusion between different parts of the transgene. As a result, Wong et al. performed droplet digital PCR to quantify the number of copies of intact Cre recombinases as one per transgene. Results revealed only one functional copy of intact Cre present in hemizygous mice. This finding may explain why - when crossed with a mouse having a floxed gene - there may be incomplete ablation or reconstitution in expression of the gene of interest7,8.
Although complete adipose tissue-specific excision of floxed sequences is more likely in homozygous mice (due to potentially higher Cre expression), these mice are also more susceptible to a greater reduction in Tbx18 expression. If proven to be true, future studies should also investigate whether this might have any ramifications on proper embryonic development.
Importance of thorough genetic characterization in engineered animal models
Thorough characterization of genetically engineered animals allow for better correlation between transgene expression and the observed phenotypes. Indeed, any unexpected genetic variations - accompanying genetic manipulations – can potentially disrupt the regulatory or coding region of an essential endogenous gene, thereby confounding results and interpretation of the data. With a single primer pair, TLA technology can – in a reliable and cost-effective manner - uncover any (trans)genes of interest as well as sequence neighboring regions (up to hundreds of kilobases). By generating very high sequencing coverage across the region of interest, TLA will help determine integration site(s) and will enable the robust detection of all genetic variants (including structural variations) in and around genes of interest.
For more publications on TLA for genetic characterization of transgenic animal models, visit our publications page.
 B6.FVB-Tg(Adipoq-cre)1Evdr/J Stock No: 028020, Adipoq-Cre. The Jackson Laboratory; 2020 [cited 2020 Sep 5]. Available from: https://www.jax.org/strain/028020
 Eguchi J, Wang X, Yu S, et al. Transcriptional control of adipose lipid handling by IRF4. Cell Metab. 2011;13:249–259.
 Wong AM , Patel TP, Altman EK, Tugarinov N, Trivellin G & Yanovski JA. (2021). Characterization of the adiponectin promoter + Cre recombinase insertion in the Tg(Adipoq-cre)1Evdr mouse by targeted locus amplification and droplet digital PCR, Adipocyte, 10:1, 21-27, DOI: 10.1080/21623945.2020.1861728
 Bussen M, Petry M, Schuster-Gossler K, et al. The T-box transcription factor Tbx18 maintains the separation of anterior and posterior somite compartments. Genes Dev. 2004;18:1209–1221.
 Wu SP, Dong XR, Regan JN, et al. Tbx18 regulates development of the epicardium and coronary vessels. Dev Biol. 2013;383:307–320.
 Wiese C, Grieskamp T, Airik R, et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res. 2009;104:388–397.
 Pereira S, O’Dwyer SM, Webber TD, et al. Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues. Sci Rep. 2019;9:3307.