Marked repression of CFTR mRNA in the transgenic Cftr^tm1kth mouse model

Primary nasal septal epithelia cultures allow mechanistic comparisons that correspond well with the in vivo nasal potential difference assay, a mainstay of cystic fibrosis transmembrane conductance regulator detection in vivo, and have been viewed as suitable models for testing the activity of CFTR correctors and potentiators in “personalized medicine” type approaches to cystic fibrosis (CF) therapeutics [1–3]. Primary nasal septal epithelia from mice (MNSE) have substantially greater CFTR activity in Ussing chamber analysis than comparably processed tracheal monolayers, and allow direct analysis of otherwise genetically identical (congenic) animals without the confounding effects attributable to mixed genetic backgrounds [4].

There are three widely utilized F508del transgenic mouse models—Cftr^tm1Kth (Utah), Cftr^tm2Cam (Rotterdam), and Cftr^tm1Eur (Cambridge). The Cftr^tm1Kth mutated allele contains a 3-bp (CTT) deletion between bases 1656 and 1660, which results in a loss of a phenylalanine residue in exon 10 at a position which corresponds to human position 508 [5]. This exon 10 replacement strategy left a selective marker gene inserted in one of the introns. This was a similar strategy to the creation of the Cftr^tm2Cam mouse model [6]. This marker gene is likely to have an effect on transcription activity or mRNA levels of the mutant allele, but does not affect the protein product. The other widely utilized F508del mouse model is Cftr^tm1Eur, which was made with a “hit and run” procedure that resulted in a mutant exon from which transcriptional activity of the mutant allele is identical to the normal allele [7]. While the Cftr^tm2Cam mouse was noted to have 1/3 of the transcriptional activity in all tissues tested, prior studies concerning Cftr^tm1Kth transgenic mice have indicated tissue specific differences in CFTR expression—specifically lower levels of F508del CFTR mRNA in the upper intestine compared to wild type animals. Normal levels of CFTR mRNA were reported previously in F508del murine lung, testes, and submaxillary glands in the same strain [5]. Given the high interest in evaluating misprocessing correctors of F508del CFTR in a relevant in vivo and in vitro model of disease, proper F508del CFTR expression is critical to promote maturation of a similar pool of misprocessed protein.

After the University of Alabama at Birmingham Institutional and Animal Care and Use Committee approval, MNSE tissues from wild type (C57BL/6), heterozygous F508del, and homozygous F508del/F508del transgenic (Cftr^tm1Kth) were cultured on permeable supports for 14 days to confluence and full differentiation. This F508del mouse strain was obtained from Jackson Labs (stock # 002515) and backcrossed to the C57BL/6 line 6 times. The mice are greater than 98% on the BL/6 background. Quantitative RT-PCR analysis (reported as relative mRNA ± S.D.) showed dramatic differences in CFTR mRNA among homozygous, heterozygous, and wild type both in MNSE cultures and tissues (Fig. 1A). Whether these differences are mediated by higher expressivity in a pure epithelial cell population (compared to mixed cells from native tissues), decreased CFTR transcription, or message instability will require further investigation. In addition, there was minimal F508del CFTR-mediated anion transport following 24 hour low-temperature correction (27 °C) in Ussing chambers (Fig. 1B).

Fig. 1.

Relative CFTR mRNA expression in wild type, F508del heterozygous, and homozygous murine nasal tissues and cell cultures (A). CFTR mRNA expression in F508del homozygous murine nasal tissues (n = 6 per condition) and cultures (n = 9 per condition) were significantly reduced (p < 0.05) when compared to heterozygous (tissue: 2.7 ± 0.3 fold, cell culture: 10.6 ± 3.6) and wild type (tissue: 5.7 ± 1.2, cell culture: 20.0 ± 8.7). Representative Ussing chamber tracings of wild type, F508del/F508del, and temperature corrected (TC) F508del/F508del cell cultures (B). After blockade of epithelial sodium channels with amiloride (100 μM), temperature corrected cells (24 h at 27 °C incubation) reveal minimal “corrected” F508del CFTR activity in response to the adenylyl cyclase agonist forskolin (20 μM). CFTR(inh)172 (10 μM) blocks CFTR-mediated anion transport.

Our findings suggest significant limitations of nasal airway cultures from Cftr^tm1Kth mice, and previous studies of F508del CFTR from the ^{tm1 Kth} background should be re-evaluated in this light [8]. For example, potentiator activation profiles observed in F508del CF mice may underestimate potency as a result of diminished levels of mRNA in this murine genotype. While murine CFTR is insensitive to many agents that enhance the activity of human CFTR (e.g. ivacaftor) [9], agents (e.g. flavonoids) known to increase channel opening in both murine and human CFTR should be appraised in this light. Furthermore, this mouse model should not be utilized for evaluating F508del misprocessing correctors with upper airway experiments in vivo (e.g. NPD measurements) or in vitro (primary nasal cell culture electrophysiology) endpoints. The findings also provide a cautionary note indicating the potential for highly discordant levels of CFTR mRNA from certain CFTR genotypes should nasal cells be developed as a universal screen meant to “personalize” CFTR modulation therapy in patients with rare genetic defects.