RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function

The Cre/loxP recombinase system for performing conditional gene targeting experiments has been very useful in exploring genetic pathways that control both the development and function of pancreatic beta-cells. One particular line of transgenic mice (B6.Cg-Tg(Ins2-cre)25Mgn/J), commonly called RIP-Cre, in which expression of Cre recombinase is controlled by a short fragment of the rat insulin II gene promoter has been used in at least 21 studies on at least 17 genes. In most of these studies inactivation of the gene of interest was associated with either glucose intolerance or frank diabetes. Experimental evidence has been gradually emerging to suggest that RIP-Cre mice alone display glucose intolerance. In this study experiments from three laboratories demonstrate that RIP-Cre mice, in the absence of genes targeted by loxP sites, are glucose intolerant, possibly due to impaired insulin secretion. In addition, we review the use of RIP-Cre mice and discuss possible molecular underpinnings and ramifications of our findings.     

Alterations of pancreatic beta-cell mass and islet number due to Ins2-controlled expression of Cre recombinase: RIP-Cre revisited; part 2.

Tissue-specific disruption of genes by targeted expression of Cre recombinase in insulin-producing cells has been widely used to explore pathways involved in regulation of pancreatic beta-cell mass. One particular line of transgenic mice [B6.Cg-Tg(Ins2-cre)25Mgn/J], commonly called RIP-Cre, in which the expression of Cre recombinase is controlled by a short fragment of the rat insulin II gene promoter has been used on at least 20 genes in at least 27 studies. In the majority of these studies (15 out of 27) inactivation of the gene of interest was associated with alterations in islet architecture, islet mass, or pancreatic insulin content. We have tested the hypothesis that genomic integration or expression of Cre recombinase alone causes alterations of beta-cell mass by quantifying islet number and mass in RIP-Cre mice. We have observed a significant hypoplasia of beta-cells in young RIP-Cre mice, and a significant hyperplasia of islets in older RIP-Cre animals. These findings suggest that glucose intolerance and impaired insulin secretion previously described for younger RIP-Cre mice might be caused by transgene-associated islet hypoplasia, and that hyperplasia in older mice might reflect a compensatory response to transgene-related glucose intolerance.     

Rearrangement of a conditional allele regardless of inheritance of a Cre recombinase transgene

To generate conditional gene knockouts in osteoblasts, we previously developed transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively). Col-Cre mice were bred with mice in which exon 4 of the Igf1 gene is flanked by loxP sites. Mating units were arranged such that either the male or the female breeder transmitted the Col-Cre transgenes. Progeny were evaluated for Cre-mediated Igf1 gene rearrangement. We found that the loxP-flanked Igf1 locus was rearranged in the absence of inheritance of the Cre transgene. The incidence was 50 and 28% with Col2.3-Cre and Col3.6-Cre females, respectively, and 15 and 18% with Col2.3-Cre and Col3.6-Cre males, respectively.     

肥大软骨细胞特异性表达Cre重组酶转基因小鼠的建立

肥大软骨细胞是软骨细胞的终末分化形式,在软骨内成骨过程中发挥十分关键的作用。为了研究肥大软骨细胞在骨骼发育过程中的功能,我们构建了在8.2 kb小鼠X型胶原基因（Col10a1）启动子控制下表达Cre重组酶的转基因小鼠品系（Col10a1-8.2-Cre）。采用显微注射法将11.5 kb的转基因片段引入小鼠基因组,共注射受精卵328枚,获得子代鼠51只,经PCR基因型鉴定有3只在基因组上整合有Cre重组酶基因。PCR检测发现Col10a1-8.2-Cre转基因在含有肥大软骨细胞的组织中表达。为了检测Cre重组酶表达的强度和组织特异性,转基因小鼠与ROSA26报告小鼠交配。子代ROSA26;Col10a1-8.2-Cre双转基因小鼠LacZ染色检测的结果显示,Cre重组酶在所有的肥大软骨细胞中表达。原位杂交的结果验证Col10a1-8.2-Cre转基因表达在肥大区的上端。以上结果表明,我们建立的肥大软骨细胞特异性表达Cre重组酶的转基因小鼠品系可以作为一种遗传学工具,介导目的基因在肥大软骨细胞中的敲除。     

Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis.

To elucidate the biological role of Stat3 in the skin, conditional gene targeting using the Cre-loxP system was performed as germline Stat3 ablation leads to embryonic lethality. K5-Cre;Stat3(flox/-) transgenic mice, whose epidermal and follicular keratinocytes lack functional Stat3, were viable and the development of epidermis and hair follicles appeared normal. However, hair cycle and wound healing processes were severely compromised. Furthermore, mutant mice expressed sparse hair and developed spontaneously occurring ulcers with age. Growth factor-dependent in vitro migration of Stat3-disrupted keratinocytes was impaired despite normal proliferative responses. We therefore conclude that Stat3 plays a crucial role in transducing a signal required for migration but not for proliferation of keratinocytes, and that Stat3 is essential for skin remodeling, including hair cycle and wound healing.     

Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine β-lactoglobulin gene that confer locus commitment to heterogeneous tissues

In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine -lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus     

角质细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有角质细胞特异性角质素5启动子、Cre重组酶基因和人生长激素基因plyA的转基因载体pK5-Cre-hGH。以显微注射的方法将4．2kb的转基因片段K5-Cre-hGH引入小鼠基因组,共注射720枚受精卵,其中龄5枚移植至29只假孕母鼠的输卵管中发育,获得子代小鼠48只,经基因型鉴定有12只小鼠在其基因组上整合有Cre基因,整合率为25％。将带有cre重组酶基因的小鼠与基因组上携带loxP位点的smad4条件基因打靶小鼠杂交以检测Cre重组酶组织特异性表达情况以及介导重组的功能。结果表明,K5-Cre转基因小鼠只在皮肤组织中表达Cre重组酶并能在体内成功地介导loxP位点的重组。     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Dual embryonic origin of the mammalian enteric nervous system

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.     

Ectopic expression of chloramphenicol acetyltransferase (CAT) in the cerebellum in mice transgenic for a carbonic anhydrase II promoter-CAT construct that is without apparent phenotypic effect

We have developed six transgenic lines of mice with constructs containing presumptive 5' regulatory regions of carbonic anhydrase II (CA II). Four of the lines contained 1,100 bases of the 5' flanking region of the human CA II gene, and two transgenic lines resulted from a construct containing 500 bases of the 5' flanking region of the mouse CA II gene. Tissue-specific expression of the chloramphenicol acetyltransferase (CAT) gene was not obtained in any of the transgenic lines. One of the transgenic lines was found to have high levels of expression of CAT in cerebellum. This expression persisted through multiple generations and was independent of the parental origin of the transgene. On the assumption that the expression was due to the insertion of the transgene in or near a gene expressed normally in cerebellum, homozygous mice were bred for the transgenic insert to see if a mutation might have been induced. Homozygous mice were found and seemed to be normal in all aspects of their phenotype studied. Thus, in this case, neither the insertion of the gene nor the ectopic expression of CAT seemed to be harmful to the animals.