Directing the IGF1R overexpression and the Igf1r knockout to the intestinal

In order to investigate the role of the IGF1R during intestinal development and maintenance as well as during intestinal tumor formation and progression in vivo, we wanted to specifically overexpress the human IGF1R and to knockout endogenous Igf1r, respectively, in the murine intestinal epithelium. For this purpose a suitable promoter had to be selected. To date, several promoters that guide transgene expression in the intestine are known. In the following chapter the different established mouse lines in which transgene expression is under the control of an intestine-specific promoter will be discussed:

Pinto et al. (1999) reported that a 9 kb regulatory region of the mouse villin gene is sufficient to direct high and tissue-specific transgene expression in epithelial cells along the crypt-villus axis in the small intestine and colon. Additionally, this region was found to maintain a gradient of transgene expression from the crypts to the tips of the villi that is precisely similar to that exhibited by the murine villin gene (Pinto et al. 1999).

Madison (2002) generated a 12.4 kb variant of the regulatory region of the villin gene that was proven to drive high expression of two reporter genes within the entire intestinal epithelium. Both the 9 kb and the 12.4 kb promoter variants have been successfully used to generate several intestine-specific transgenic mouse models (Roth et al. 2009). El Marjou et al. (2004) generated a mouse expressing the tamoxifen-dependent cre recombinase under the 9 kb villin promoter construct and thus provided an inducible model for the tissue- and time-specific cre recombinase-mediated recombination in the intestinal epithelium. Interestingly, the recombined locus lasted, despite rapid and continuous intestinal renewal, for 60 days after tamoxifen administration, indicating that epithelial progenitor cells have been targeted (El Marjou et al. 2004). Roth et al. (2009) generated a transgenic doxycycline-inducible mouse model that expresses the rtTA2-M2 under the control of the 12.4 kb villin promoter, and proved transgene expression throughout the entire intestine from the pyloric area to the distal colon and rectum. Furthermore, expression was observed in all epithelial cells of the small intestine and colon, with decreased levels in the crypts of the small intestine and the lower half of colonic crypts, respectively (Roth et al. 2009). The advantage of the villin promoter is due to the fact that transgene expression is restricted to epithelial cells throughout the intestine. Only a small amount of villin expression was found in the proximal tubules of the kidney. However, the big disadvantage of the villin

promoter is the finding that transgene expression is not limited to epithelial cells of the colon, but was also observed in epithelial cells of the small intestine and in the kidney.

For this reason several attempts have been performed to generate a model with a more limited expression pattern (Johnson and Fleet 2013):

Saam and Gordon (1999) generated a mouse model that expresses the rtTA under the control of transcriptional regulatory elements from the fatty acid-binding protein gene (Fabp) as well as the cre recombinase under the control of tet operator sequences.

This mouse model allowed the induction of the recombination only in the intestine in the presence of doxycycline. Upon doxycycline administration, recombination occurred in the small intestinal, cecal and colonic epithelium. The recombined locus was shown to persist for at least 60 days after doxycycline withdrawal, indicating that recombination has occurred in the intestinal epithelial progenitor cells (Saam and Gordon 1999).

Means et al. (2008) generated the so called K19^CreERT mouse in which the tamoxifen-dependent CreER^T was knocked into the endogenous cytokeratin 19 locus. K19 is known to be highly expressed in the ducts of the adult pancreas and of the liver. The K19^CreERT mouse revealed recombination in epithelial cells of pancreatic and hepatic ducts, the stomach and the intestine (Means et al. 2008).

Barker et al. (2007) generated a tamoxifen-inducible mouse in which expression of the lacZ reporter was under the control of Lgr5-expressing cells. Therefore, they integrated the enhanced green fluorescent protein (EGFP)-IRES-creERT2 cassette into the first

exon of the Lgr5 gene. Subsequently, they crossed the EGFP-IRES-creERT2 knock-in allele with the cre-activatable Rosa26-lacZ reporter

strain. Thus, by injecting tamoxifen the CreER^T2 fusion protein was activated in Lgr5-expressing cells. They found lacZ expression at the typical CBCC position, indicating that Lgr5-positive cells are the small intestinal and colonic stem cells (Barker et al. 2007). However, Lgr5 was further found to be expressed in e.g. hair follicle and mammary glands and is therefore a more general marker of adult stem cells rather than an intestine-specific stem cell marker (Barker et al. 2007; Johnson and Fleet 2013).

Hinoi et al. (2007) generated a mouse that drives cre recombinase expression under the CDX2P-NLS promoter and that carries a loxP-modified Apc allele. The human CDX2 homeobox gene is known to be expressed in epithelial cells throughout the adult

small intestine and colon. They found that mice carrying the

CDX2P-NLS Cre recombinase transgene and a floxed Apc allele develop colorectal adenomas and even carcinomas in approx. 15 to 20% of mice. Unfortunately, they observed β-gal reporter expression in the tail bud and caudal part of the neural tube in CDX2P-NLS Cre recombinase transgenic embryos, whereas transgene expression was limited to distal small intestine, cecum, colon and rectum in adult mice.

Furthermore, they also found tumor development in the small intestine and predicted that the small intestinal tumor burden inhibits the development and progression of colorectal tumors in the mouse (Hinoi et al. 2007).

Xue et al. (2010) generated a transgenic mouse in which cre recombinase expression is restricted to epithelial cells of the colon. Therefore, a 10.6 kb colon-specific promoter and a 2.5 kb erythroid / colon enhancer fragment of the mouse carbonic anhydrase 1 (mCA1) gene, which is conserved between mouse and human, were used to drive cre recombinase expression (CAC). They found transgene expression limited to epithelial

cells of the colon. Afterwards, the CAC mice were crossed with APC^580S mice (see chapter 4.5.3), which carry loxP sites flanking the introns 13 and 14 of the Apc gene, to generate heterozygous (CAC;APC^580S/+) and homozygous

(CAC;APC^580S/580S) Apc knockout mice, and observed adenomas predominantly in the distal part of the colon. However, carcinomas were not detected in this mouse model (Xue et al. 2010).

In the present study, the villin promoter was chosen to drive intestinal-specific IGF1R overexpression and the Igf1r knockout, respectively. As previously described, several intestine-specific promoters are known, leading to the question if the villin promoter is the accurate promoter to drive transgene expression in the present study.

The main advantage of the villin promoter is the fact that it is known to drive stable and homogeneous expression of transgenes in all epithelial cells of the small intestine and colon along the crypt-villus axis (El Marjou et al. 2004). However, this advantage could be interpreted at the same time as a disadvantage since tumor development occurs predominantly in the distal colon and rectum in human sporadic CRC and familial adenomatous polyposis (FAP). In addition, the villin promoter was found to be active in the putative undifferentiated stem cell compartment of the intestinal epithelium (El Marjou et al. 2004; Roth et al. 2009). This fact is a big advantage of the villin promoter, because it was shown that villin-guided transgene expression thus lasted for more than 60 days (El Marjou et al. 2004) despite the remarkable constant and rapid renewal capacity of intestinal epithelial cells (Bustos-Fernández 1983; Medema and

Vermeulen 2011). If transgene expression is directed by a promoter that is only active in differentiating epithelial cells in the intestine, the recombination efficacy could become too low due to the loss of differentiating epithelial cells during intestinal renewal (El Marjou et al. 2004). A general disadvantage of the villin promoter is the fact that it also drives transgene expression in the proximal tubules of the kidney (Roth et al. 2009), indicating that the villin promoter is not intestine-specific. In addition, villin-guided transgene expression was observed to be stronger in the differentiated cells compared to cells of the crypts in the small intestine and cells of the lower half of the crypts in the colon (El Marjou et al. 2004; Roth et al. 2009). Regarding the present study, this finding is a big disadvantage of the villin promoter since endogenous Igf1r reveals an opposing expression pattern with high expression in the crypts of the small intestine and at the bottom of the crypts of the colon and low expression in the villi of the small intestine and at the tip of the crypts of the colon (see chapter 4.3). These opposing expression gradients of villin and endogenous Igf1r lead to the assumption that the Igf1r knockout may be impaired in the crypts because villin expression is low (for detailed discussion see chapter 4.5.2). Furthermore, also IGF1R overexpression is suggested to be not optimal, because villin-guided ectopic IGF1R is predominantly

expressed in the villi of the small intestine and at the tip of the colonic crypts (see chapter 3.6), whereas endogenous Igf1r is mainly expressed in the crypts of the

small intestine and at the bottom of the colonic crypts (see chapter 4.3). Thus, villin-guided ectopic IGF1R expression reveals a different localization compared to endogenous Igf1r expression, which might have influenced the results of the present study.

Comparison of the villin promoter with the other described promoters shows that also the other promoters exhibit advantages and disadvantages. The main advantages and disadvantages of the different promoters are summarized in table 2. The advantage of the Fabp promoter is that it also targets the progenitor cells of the intestine, leading to recombination that lasts for more than 60 days, which was also shown for the villin promoter. The disadvantage of the Fabp promoter is that it drives transgene expression in the small intestine, cecum and colon (Saam and Gordon 1999). Transgene expression under control of the CDX2P-NLS promoter is found in epithelial cells throughout the adult small intestine and colon, which is also true for the villin promoter.

Interestingly, CDX2P-NLS Cre (CPC) mice and Villin-Cre mice were crossed with mice carrying loxP-modified Apc alleles (Apc^loxP/loxP) and tumor development was compared

between these two mouse lines. CPC;Apc mice developed 5 to 8 tumors per mouse in the colon and 3 tumors per mouse in the small intestine, whereas Vilin-Cre;Apc mice revealed formation of approx. 36 tumors per mouse, but of which the vast majority developed in the small intestine (Hinoi et al. 2007). On the one hand, this comparison shows that the CDX2P-NLS promoter seems to be the better choice, because the majority of tumors developed in the colon. On the other hand, the villin promoter appears to be the superior choice, because the incidence of tumor formation is much higher than under the control of the CDX2P-NLS promoter. The Lgr5 promoter drives transgene expression in the Lgr5-positive CBC stem cells. However, Lgr5 is also expressed in hair follicle and mammary glands, and thus, Lgr5 is more a general marker of adult stem cells rather than an intestine-specific stem cell marker (Barker et al. 2007). Transgene expression under the control of the mCA1 promoter was found to be limited to epithelial cells of the colon (Xue et al. 2010) which is a big advantage over the villin promoter. Furthermore, mCA1-guided β-galactosidase expression was found to reach from the crypt base to the luminal surface (Xue et al. 2010) which is more consistent with the expression pattern of endogenous Igf1r than villin expression. This comparison leads to the suggestion that the mCA1 promoter could be a better choice to drive transgene expression than the villin promoter in the present study. Of note, a mouse model that expresses a reverse tetracycline transactivator under the control of the mCA1 promoter does not exist to date. Thus, for the present study a mouse for the mCA1-guided overexpression of the IGF1R had to be generated first.

Table 2: Summary of the advantages and disadvantages of the different intestine-specific promoters.

Promoter Advantage Disadvantage Reference

Villin villin-guided transgene intestine and in lower half of colonic crypts

El Marjou et al.

2004

Roth et al.

2009

mouse with villin-guided

Fabp is active in progenitor cells Fabp-guided transgene expression in small intestine, cecum and colon

Saam and Gordon 1999

Lgr5 is active in CBCCs Lgr5-guided transgene expression also in hair

Taken together, the promoters described above exhibit advantages and disadvantages over the villin promoter. In regard to choose the right promoter the work of Janssen et al. (2002) was of high interest. They generated a transgenic mouse line expressing K-ras^V12G, which is the most frequent point mutation in human tumors, under the control of the 9 kb murine villin promoter variant. At nine months of age more than 80% of the transgenic mice had developed tubuloglandular adenomas and malignant

adenocarcinomas predominantly in the small intestine (90%), but also in the colon (7%) (Janssen et al. 2002). However, Cheung et al. (2010) generated a mouse line with a cre-regulable null allele of the Apc gene and crossed these Apc^fle1-15 mice to Villin-Cre transgenic mice. At approx. 4 months of age, the Apc^+/fle1-15; Villin-Cre mice developed several adenomas in their small intestines and colons. However, carcinomas were not detected (Cheung et al. 2010). These examples in which transgene expression guided by the villin promoter induced intestinal tumor formation further indicate that the villin promoter was a suitable promoter for the present study. Since the Villin-rtTA2-M2 mouse line expressing the reverse tetracycline transactivator and the Villin-CreER^T2 mouse line expressing the cre recombinase under the control of the villin promoter, respectively, were available, we decided to utilize these mouse lines for the present study to generate the IGF1R overexpressing and Igf1r knockout mice.