Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter–Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method

Raman, Priyadarshini; Grachtchouk, Vladimir; Lyons, Robert H.; Koenig, Ronald J.

doi:10.1089/thy.2015.0215

Cited by 4 publications

(3 citation statements)

References 12 publications

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“…Unfortunately, although identifying insertion site is straightforward for invertebrates, available methods for mice (Burgess et al, 1995;Sharpe et. al, 1999;Suzuki et al, 2006;Sha et al, 2007;Liang et al, 2008;Dubose et al, 2013;Srivastava et al, 2014;Raman et al, 2015) have been cumbersome, little used and, in our hands, largely unsuccessful. Recently, however, a newly developed method termed Targeted Locus Amplification (TLA) was introduced that appeared to be more promising (de Vree et al, 2014;Cain-Hom et al, 2017).…”

Section: Introductionmentioning

confidence: 91%

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Laboulaye

Duan

Qiao

et al. 2018

Preprint

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Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other proteins. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, and sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other's expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

show abstract

Section: Introductionmentioning

confidence: 91%

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Laboulaye

Duan

Qiao

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…We reasoned that if this were a general phenomenon, the endogenous gene might play a role in the development or function of the marked cells. Unfortunately, although identifying insertion site is straightforward for invertebrates, available methods for mice ( Burgess et al, 1995 ; Sharpe et al, 1999 ; Suzuki et al, 2006 ; Sha et al, 2007 ; Liang et al, 2008 ; Dubose et al, 2013 ; Srivastava et al, 2014 ; Raman et al, 2015 ) have been cumbersome, little used and, in our hands, largely unsuccessful. Recently, however, a newly developed method termed Targeted Locus Amplification (TLA) was introduced that appeared to be more promising ( de Vree et al, 2014 ; Cain-Hom et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Laboulaye

Duan

Qiao

et al. 2018

Front. Mol. Neurosci.

View full text Add to dashboard Cite

Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other protein. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, it is sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other’s expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

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“…TG cog/cog mice (C57BL6/J) were obtained from The Jackson Laboratory. Hrd1 -floxed mice ( 54 ), Atg7 -floxed mice ( 55 ), and Tpo Cre mice ( 56 , 57 ) were used as previously described. Tpo Cre mice were back-bred with Hrd1 fl/fl or Atg7 fl/fl mice to generate thyrocyte-specific Hrd1 TPO and Atg7 TPO mice, respectively.…”

Section: Methodsmentioning

confidence: 99%

Perturbation of endoplasmic reticulum proteostasis triggers tissue injury in the thyroid gland

Zhang,

Young,

Liao

et al. 2023

JCI Insight

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Defects in endoplasmic reticulum (ER) proteostasis have been linked to diseases in multiple organ systems. Here we examined the impact of perturbation of ER proteostasis in mice bearing thyrocyte-specific knockout of either HRD1 (to disable ER-associated protein degradation [ERAD]) or ATG7 (to disable autophagy) in the absence or presence of heterozygous expression of misfolded mutant thyroglobulin (the most highly expressed thyroid gene product, synthesized in the ER). Misfolding-inducing thyroglobulin mutations are common in humans but are said to yield only autosomal-recessive disease — perhaps because misfolded thyroglobulin protein might undergo disposal by ERAD or ER macroautophagy. We find that as single defects, neither ERAD, nor autophagy, nor heterozygous thyroglobulin misfolding altered circulating thyroxine levels, and neither defective ERAD nor defective autophagy caused any gross morphological change in an otherwise WT thyroid gland. However, heterozygous expression of misfolded thyroglobulin itself triggered significant ER stress and individual thyrocyte death while maintaining integrity of the surrounding thyroid epithelium. In this context, deficiency of ERAD (but not autophagy) resulted in patchy whole-follicle death with follicular collapse and degeneration, accompanied by infiltration of bone marrow–derived macrophages. Perturbation of thyrocyte ER proteostasis is thus a risk factor for both cell death and follicular demise.

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Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter–Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method

Cited by 4 publications

References 12 publications

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

Perturbation of endoplasmic reticulum proteostasis triggers tissue injury in the thyroid gland

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