利用Cre/LoxP系统删除转基因山羊体内的选择标记基因

在制备转基因家畜过程中的一个关键步骤是使用选择标记基因 (Selectable marker genes,SMGs) 将转基因整合细胞从大量的正常细胞中筛选出来,这导致了SMGs整合入家畜的基因组内持续传递给后代。SMGs已被证明能够显著影响基因组内整合位点处的基因调控,也增加了对转基因动物安全评价的复杂性。为了确定转基因山羊制备过程中SMGs的删除时机和删除方法,在体细胞克隆前后两个时段内,利用Cre/loxP系统删除SMGs的可行性,同时比较了蛋白转导和质粒共转染两种Cre导入方式的删除效率。结果表明：尽管在首次对山羊成纤维细胞进行遗传修饰后即可进行SMGs删除,但两次遗传修饰导致细胞严重老化,无法用于后续的体细胞克隆羊制备。在转基因山羊的成体细胞中删除SMGs不存在上述问题,成功率高,缺点是试验周期长、耗资增大。Cre表达质粒瞬时转染能够删除SMGs,但有超过30%的无SMGs细胞克隆中整合有质粒序列。TAT-CRE蛋白质转导方法可以避免引入的新外源基因,SMGs删除率达到43.9%~72.8%,是一种较佳的SMGs删除手段。     

Conditional gene recombination by adenovirus-driven Cre in the mouse uterus

Cre-mediated conditional gene targeting has been shown to be successful in many cell and tissue types. However, gene recombination in the uterus with heterogeneous cell types by Cre activation is not yet well established. Using recombinant adenoviruses expressing a functional Cre (ADV-Cre) and ROSA26 reporter mice, we show here that ADV-Cre infused intraluminally in a small volume (10 microl) conditionally excises the loxP site, resulting in lacZ expression in uterine luminal epithelial cells without significantly affecting pregnancy. In contrast, a similar intraluminal infusion of ADV-Cre in a larger volume (50 microl) damages the normal architecture and integrity of the luminal epithelium, inducing gene recombination in the underneath stromal cells, with disruption of pregnancy. Further, decidualizing stromal cells at the implantation sites can be targeted by ADV-Cre after intravenous administration on days 5-6. This route of administration also elicits Cre activity in other tissues, including the liver, spleen, ovary, and, more remarkably, in the adrenal cortex. These findings demonstrate the feasibility of achieving conditional expression or deletion of specific genes in uterine cells at desired times and physiological states.     

Cre重组酶表达载体的构建及其在转基因小鼠胰腺中的表达

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性条件敲除研究的关键。采用PCR扩增大鼠胰岛素基因705bp启动子指导发胰岛细胞中特异表达;同时采用改构的Cre重组酶基因,在其5'端添加有真核核糖体结合序列和核定位序列使Cre重组酶能穿越核膜在细胞核能发挥功能;同时,为了保证原核基因Cre能在真核系统顺利表达,在其3'端添加含内含子的人生长激素基因。构建的表达载体在去除原核序列后用显微注射方法转基因小鼠,在出生的27只仔鼠中,PCR检测共获得7只Cre整合阳性的转基因小鼠,整合率26％。这种Cre转基因小鼠与基因组小携带LoxP位点的条件基因打靶小鼠交配,在胰腺组织中可以检测到Cre介导的重组,表明Cre在转基因小鼠胰腺中有表达。     

Male germline recombination of a conditional allele by the widely used Dermo1‐cre (Twist2‐cre) transgene

Conditional gene knockout using the Cre/loxP system is instrumental in advancing our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature that recombination mediated by some Cre transgenes can occur in unexpected tissues. Dermo1‐Cre (Twist2‐Cre) has been widely used to target skeletal lineage cells as well as other mesoderm‐derived cells. Here we report that Dermo1‐Cre exhibits spontaneous male germline recombination activity leading to a Cre‐mediated recombination of a floxed Ptk2 (Protein tyrosine kinase 2, also known as Fak [Focal adhesion kinase]) allele but not a floxed Rb1cc1 (RB1 inducible coiled‐coil 1, also known as Fip200 [FAK‐family Interacting Protein of 200 kDa]) allele at high frequency. This ectopic germline activity of Dermo1‐Cre occurred in all or none manner in a given litter. We demonstrated that the occurrence of germline recombination activity of Dermo1‐Cre transgene can be avoided by using female mice as parental Dermo1‐Cre carriers.     

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (Vγ^LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (Vγ^Δ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the Vγ^Δ locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined Vγ^LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the Vγ^LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997. © 1997 Wiley-Liss, Inc.     

Germline transmission and efficient DNA recombination in mouse embryonic stem cells mediated by adenoviral-Cre transduction

Following gene targeting, a loxP-neo-loxP cassette was introduced into ES cells. The presence of a selectable marker such as neo in the targeted allele may result in gene interference in flox mice or unexpected phenotypes due to genetic ambiguity in direct knockout mice. Typically, the neo cassette is selectively removed by transient expression of the Cre recombinase in targeted ES cell. However, this method involves a tedious process of selecting, expanding, and screening ES cell clones which may compromise germline competency. Here, we describe a novel method of combining adenovirus-Cre mediated gene recombination with ES gene targeting to facilitate efficient loxP-neo-loxP removal in ES cells. We demonstrate that adenovirus-Cre infected ES cells can retain their germline competency. The procedures described here facilitate a rapid genetic manipulation of ES cells to obtain neo-free knockout animals, multiple gene targeting, homozygous mutant ES cells ideal for in vitro characterization, or Rag-deficient blastocyst complementation.     

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte‐specific recombination of loxP‐flanked (“floxed”) alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two‐color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre‐dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development. genesis 47:789–792, 2009. © 2009 Wiley‐Liss, Inc.     

Cre/LoxP重组系统的改造及在树干毕赤酵母中的应用

为了实现在P.stipitis中进行无痕基因敲除,以Cre/LoxP系统为研究对象,首先通过同源重组构建尿嘧啶营养缺陷型树干毕赤酵母(ura3-);同时通过定点突变pSH47-Hpt质粒的hpt基因和cre基因,将CDS区CTG突变为TTG;最后以乙醛脱氢酶基因为靶基因,验证突变后的Cre/LoxP系统在P.stipitis进行无痕基因敲除的可行性。结果表明:本文在P.stipitis中成功使用潮霉素B抗性标记,经过修饰后的Cre/LoxP敲除系统能够在P.stipitis中无痕敲除目的基因,为后续研究P.stipitis功能基因和改造代谢途径提供了一种试验方法和筛选标记。     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

Generation of a germ cell nuclear factor conditional allele in mice

Two recombination steps in embryonic stem (ES) cells were adopted to generate a floxed Germ Cell Nuclear Factor (GCNF) allele. First, a targeting vector containing a loxP site upstream of exon 4, encoding the DNA binding domain (DBD), and a floxed NeoTK double selection cassette downstream of exon 4 was integrated into the GCNF locus by homologous recombination. Second, a Cre-expressing vector was transiently introduced to remove the floxed NeoTK cassette via site-specific recombination. Heterogeneous ES cell populations were found in a single colony after Cre transfection and were separated using an ES cell re-pick step. Floxed GCNF mice were generated and had normal GCNF expression in the adult gonads. Using the Msx2Cre transgenic mice, the floxed GCNF can be completely deleted in the female germline. Taken together, the floxed GCNF mice were successfully generated and female germline deletion of the floxed GCNF allele was achieved using Msx2Cre mice.     

Different Cre systems induce differential microRNA landscapes and abnormalities in the female reproductive tracts of Dgcr8 conditional knockout mice

ObjectivesThe female reproductive tract comprises several different cell types. Using three representative Cre systems, we comparatively analysed the phenotypes of Dgcr8 conditional knockout (cKO) mice to understand the function of Dgcr8, involved in canonical microRNA biogenesis, in the female reproductive tract.Materials and Methods Dgcr8 ^f/f mice were crossed with Ltf ^icre/+, Amhr2 ^cre/+ or PR ^cre/+ mice to produce mice deficient in Dgcr8 in epithelial (Dgcr8 ^ed/ed), mesenchymal (Dgcr8 ^md/md) and all the compartments (Dgcr8 ^td/td) in the female reproductive tract. Reproductive phenotypes were evaluated in Dgcr8 cKO mice. Uteri and/or oviducts were used for small RNA‐seq, mRNA‐seq, real‐time RT‐PCR, and/or morphologic and histological analyses.Result Dgcr8 ^ed/ed mice did not exhibit any distinct defects, whereas Dgcr8 ^md/md mice showed sub‐fertility and oviductal smooth muscle deformities. Dgcr8 ^td/td mice were infertile due to anovulation and acute inflammation in the female reproductive tract and suffered from an atrophic uterus with myometrial defects. The microRNAs and mRNAs related to immune modulation and/or smooth muscle growth were systemically altered in the Dgcr8 ^td/td uterus. Expression profiles of dysregulated microRNAs and mRNAs in the Dgcr8 ^td/td uterus were different from those in other genotypes in a Cre‐dependent manner.Conclusions Dgcr8 deficiency with different Cre systems induces overlapping but distinct phenotypes as well as the profiles of microRNAs and their target mRNAs in the female reproductive tract, suggesting the importance of selecting the appropriate Cre driver to investigate the genes of interest.     

Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double‐deficient mice

The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double‐deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double‐deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose‐binding protein and Cre (MBP‐Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks. genesis 47:545–558, 2009. © 2009 Wiley‐Liss, Inc.     

Incomplete cre‐mediated excision leads to phenotypic differences between Stra8‐iCre; Mov10l1lox/lox and Stra8‐iCre; Mov10l1lox/Δ mice

In the Cre–loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre‐mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8‐codon‐improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell‐specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre‐mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre–loxp system is used for deleting genes in a specific cell lineage and the Cre; gene^lox/Δ genotype should be used to evaluate phenotypes instead of Cre; gene^lox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 51:481–490. © 2013 Wiley Periodicals, Inc.     

A Cre knock‐in mouse line on the Sickle tail locus induces recombination in the notochord and intervertebral disks

Sickle tail (Skt) was originally identified by gene trap mutagenesis in mice, and the trapped gene is highly expressed in the notochord, intervertebral discs (IVD), and mesonephros. Here, we report the generation of Skt^cre mice expressing Cre recombinase in the IVD due to target insertion of the cre gene into the Skt locus by recombinase‐mediated cassette exchange. Crossing a conditional lacZ Reporter (R26R), Cre expression from the Skt^cre allele specifically activates β‐galactosidase expression in the whole notochord from E9.5 onwards. In E15.5 Skt^cre;R26R embryos, reporter activity was detected in the nucleus pulposus and in a portion of the annulus fibrosus, resulting in expansion of Cre‐expressing cells in the adult IVD. Reporter activity was also seen in the Skt^cre;R26R mesonephros at E15.5. These results suggest that Skt^cre mice are useful for exploring the fate specification of notochordal cells and creating models for IVD‐related skeletal diseases. genesis 50:758–765, 2012. © 2012 Wiley Periodicals, Inc.     

Patched 1 conditional null allele in mice

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function.     

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1‐Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.