Inconsistency between hepatic expression and serum concentration of transthyretin in mice humanized at the transthyretin locus

Abstract

Human transthyretin (TTR) has about 110 variants, more than 90 of which are associated with human amyloidosis. Several groups have generated transgenic mice that carry various mutant TTR genes. However, formation of mouse/human TTR heterotetramers has been shown to be inhibitory to dissociation and subsequent amyloid formation. To avoid the effect of mouse Ttr and produce humanized mice carrying different TTR variants at high efficiency, we first produced a null allele in the mouse transthyretin locus using targeting vector that contained a neomycin resistance gene flanked by lox71 and loxP. Then, through Cre‐mediated recombination, we created a replacement allele that carried either a human normal (Val30) or mutant (Met30) TTR cDNA. This replacement resulted in a humanized TTR mouse with similar tissue‐specific profile of human TTR as that of the endogenous mouse Ttr gene. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice those of heterozygous mouse/human TTR (+/Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. This contradictory expression was due to unstable Val30 tetramers caused by low binding affinity to mouse retinol binding protein.

Severe ocular phenotypes in Rbp4-deficient mice in the C57BL/6 genetic background

“…16 ICR originated from the CD1 strain at the Institute of Cancer Research (Philadelphia, PA, USA). ICR is an albino mouse kept in closed colony at Clea (Tokyo, Japan; http://www.cleajapan.com/en/index.html).…”

“…The construction of targeting vectors and isolation of targeted ES cells were carried out as described previously, 16 except for the use of a 2.8-kb 5′ Rbp4 homologous region upstream of the ATG codon and a 6.7-kb 3′ Rbp4 homologous region downstream of the ATG codon ( Figure 1). …”

“…18 Targeted ES cell clones were confirmed by Southern blot analyses as described previously. 16 For detection of homologous recombination in the 5′ region, DNA from ES cells was digested with XhoI/SalI or MunI and the targeted allele was detected with a neo probe and a 5′ probe; for detection of homologous recombination in the 3′ region, AgeI/HpaI or SacII/HpaI digestion and a neo probe and 3′ probe were used, respectively (Supplementary Figure S1).…”

“…Northern Blot, Semiquantitative RT-PCR, and qRT-PCR Analyses Northern blot and semiquantitative reverse transcriptionpolymerase chain reaction (RT-PCR) analyses were carried out as described by Zhao et al 16 and Li et al, 19 respectively. The nucleotide sequence identity between the mouse and human Rbp4 coding regions is~87%.…”

Enhanced expression of human cDNA by phosphoglycerate kinase promoter-puromycin cassette in the mouse transthyretin locus

“…We previously developed an exchangeable gene targeting method (Wang et al 2008;Zhao et al 2008) based on the exchangeable gene trap method (Araki et al 1999(Araki et al , 2002Taniwaki et al 2005). Using this method, we successfully produced a null targeted allele at the mouse transthyretin (Ttr) gene locus as the first step, followed by highly efficient replacement of the phosphoglycerine kinase (PGK)-puro cassette with a human TTR gene, using the Cre-loxP system (Zhao et al 2008).…”