Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA

Abstract

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue-specific expression and regulation of CFTR function when animal models are used in gene therapy studies.

Aminoglycoside suppression of a premature stop mutation in a Cftr–/– mouse carrying a human CFTR-G542X transgene

“…However, the level of hCFTR required to reverse the intestinal pathology is still poorly defined, since previous studies that examined the ability of hCFTR to complement mouse Cftr knockouts produced conflicting results. In one study, a hCFTR cDNA was targeted to the mouse Cftr locus using a "knock-in" replacement strategy [26]. This construct was found to produce hCFTR mRNA in the epithelial cells of the crypts of Leiberkuhn and the intestinal villi at ~ 28% of normal murine Cftr expression.…”

Expression of CFTR from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in CF mice

“…It is interesting to note that in other studies, expression of the human CFTR cDNA in murine models did not restore full function. Thus Rozmahel et al, 38 used a 'knock-in' strategy to insert the human CFTR cDNA into the first exon of the murine CFTR gene. These mice showed no improvement in intestinal pathology or survival compared to CFTR null mice generated in a similar manner, although measurements of nasal PD were restored by expression of the human CFTR.…”

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

“…In this approach the gene of interest is replaced with a different functional sequence under the transcriptional control of cisand trans-acting elements that are part of the endogenous gene (Babinet and Cohen-Tannoudji 2001). The field of application for this method ranges from exchange of the gene of interest with a reporter gene (Elefanty et al 1998;Luche et al 2007) to the generation of humanized mice (Rozmahel et al 1997;Luo et al 2001), mainly by replacing loci with sequences of up to 10 kb, such as cDNA. However, the replacement of larger sequences might be interesting as well, e.g., the exchange of whole coding sequences including all introns to study the function of the introduced gene under ''physiological'' transcriptional control.…”