Universal highly efficient conditional knockout system in Leishmania,with a focus on untranscribed region preservation

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR‐Cas9 has been applied successfully to these parasites providing a robust tool to study non‐essential gene functions. Here, we have developed a versatile inducible system combining Di‐Cre recombinase and CRISPR‐Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre‐recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di‐Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed “universal” template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR‐based (molecular cloning‐free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.     

用Cre/LoxP和CRISPR基因工程技术构建猪肌肉生长抑制素双等位基因敲除细胞系

肌肉生长抑制素 (myostatin,Mstn)基因失活可引起哺乳动物的肌肉增生,但其调控机制尚不清楚,且缺乏可靠的试验材料验证Mstn相关分子通路的变化。本研究所用PK3108细胞系是在野生型PK15细胞系的基础上成功靶向敲除一条等位基因,在其靶位点敲入标记基因,敲除了204 bp的外显子3序列, LoxP锚定在其标记基因两侧。利用Cre/LoxP重组酶删除系统删除插入PK3108Mstn靶位点的标记,借助流式细胞仪和荧光蛋白甄别得到无标记的过渡型细胞系PK3108-2。将Cas9/sgRNA表达载体和供体DNA共转染PK3108-2,借助G418抗性筛选和倒置荧光显微镜挑选出仅带阳性标记的克隆L18,对其基因组进行PCR产物凝胶电泳与PCR产物测序,证明克隆L18在预设位点发生同源重组;对其蛋白质进行Western 印迹实验表明,Mstn被成功地敲除失活。综上结果证明,本研究实现了双等位基因的精准敲除,构建了Mstn双敲除梯度回复表达细胞系。本研究为揭示Mstn的作用机制提供理想的实验材料,也为双等位基因的敲除提供了可借鉴的技术路线。     

Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination

Conditional alleles containing LoxP recombination sites, in conjunction with Cre recombinase delivered by a variety of means, allows for spatial and temporal control of gene expression in mouse models. Here we describe a mouse strain in which a luciferase (Luc) cDNA, preceded by a LoxP-stop-LoxP (L-S-L) cassette, was introduced into the ubiquitously expressed ROSA26 locus. Mouse embryo fibroblasts derived from this strain expressed luciferase after Cre-mediated recombination in vitro. ROSA26 L-S-L-Luc/+ mice expressed luciferase in a diffuse or liver-restricted pattern, as determined by noninvasive, bioluminescent imaging, when crossed to transgenic mice in which Cre was under the control of a zygotically expressed (EIIA-Cre), or a liver-restricted (albumin-Cre), promoter, respectively. Organ-specific luciferase expression was also seen after intraparenchymal administration of an adenovirus encoding Cre. The ROSA26 L-S-L-Luc/+ strain should be useful for characterizing Cre mouse strains and for following the fate of cells that have undergone Cre-mediated recombination in vivo.     

ER-based double iCre fusion protein allows partial recombination in forebrain

Here we describe the generation of a new tamoxifen-inducible double Cre fusion protein generated by fusing two ERT2 domains onto both ends of the iCre recombinase (a codon improved Cre recombinase). This Cre fusion protein (ERiCreER) had a twofold increased activity in cell culture assays than the previously described MerCreMer Cre double fusion protein. ERiCreER was targeted to the brain by placing it under the control of the promoter from the CamKIIalpha gene using a 170 kb BAC. The fusion protein was detected in hippocampus, cortex, striatum, thalamus, and hypothalamus but not in cerebellum. The ERiCreER was cytoplasmatic in the absence of tamoxifen and translocated into the nucleus upon tamoxifen administration. The activity of the ERiCreER was tested in vivo by mating the CamKIIalpha ERiCreER transgenic line with mice harbouring exon 10 of the CREB gene flanked by two LoxP sites. In the absence of tamoxifen, no background activity was detected in mice older than 6 months. After tamoxifen administration, most if not all of the ERiCreER fusion protein translocated from the cytoplasm to the nucleus; however, only 5-10% of the "floxed" CREB allele was recombined. Recombination was also visualised at the cellular level by following the upregulation of the CREM protein, which corresponds precisely with CREB loss/recombination. Unlike in other tissues (Sohal et al., 2001; Tannour-Louet et al., 2002), it appears that in brain, although ERiCreER can bind tamoxifen, the Cre-recombinase cannot be fully activated.     

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.     

Transvection effects involving DNA methylation during meiosis in the mouse

High efficiencies of recombination between LoxP elements were initially recorded when the Cre recombinase was expressed in meiotic spermatocytes. However, it was unexpectedly found that LoxP recombination fell to very low values at the second generation of mice expressing Cre during meiosis. The inability of the LoxP elements to serve as recombination substrates was correlated with cytosine methylation, initially in LoxP and transgene sequences, but later extending for distances of at least several kilobases into chromosomal sequences. It also affected the allelic locus, implying a transfer of structural information between alleles similar to the transvection phenomenon described in Drosophila. Once initiated following Cre-LoxP interaction, neither cis-extension nor transvection of the methylated state required the continuous expression of Cre, as they occurred both in germinal and somatic cells and in the fraction of the offspring that had not inherited the Sycp1-Cre transgene. Therefore, these processes depend on a physiological mechanism of establishment and extension of an epigenetic state, for which they provide an experimental model.     

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.     

Inducible site-directed recombination in mouse embryonic stem cells.

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly, the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins, tamoxifen can efficiently induce Cre-mediated recombination, thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen, recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein, the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals.     

DNA重组酶Cre介导载体间基因的重组转移

DNA重组酶Cre可以识别LoxP位点,使含有LoxP位点的DNA分子发生重组:2个同向LoxP之间的DNA片段被删除,2个环状DNA分子被整合为一个大分子.基于Cre酶的这些作用特性,构建了一套载体间基因的重组转移体系,在Cre酶的作用下,gfp基因被从基因供体pTLG上切除下来,然后转移到基因受体pET-LoxP上,从而快速、简便地完成了gfp基因高效表达载体pET-gfp的构建.gfp基因在大肠杆菌BL21(DE3)中被诱导表达,使菌落产生了可视的绿色荧光.通过对荧光菌落的计数分析,比较了环状基因供体pTLG和线性基因供体pTLG对有效重组率的影响.使繁琐的传统载体构建变为简单的酶促反应,极大地简化了载体构建步骤,为Cre酶在基因克隆和亚克隆中的应用提供了很好的研究基础.     

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ER^T2). In the background of the Cre reporter mouse strain Rosa26^lacZ, we show that the fusion CreER^T2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 Wiley‐Liss, Inc.     

Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).     

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。     

Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.     

Cerebellar granule cell Cre recombinase expression

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.     

Tet-on和Cre/loxP系统双重调控表达丙型肝炎病毒NS3/4A蛋白酶三转基因小鼠的建立

目的:建立Tet-On调控系统和Cre/loxP基因剔除系统双重调控表达丙型肝炎病毒(HCV)NS3/4A丝氨酸蛋白酶三转基因小鼠。方法:选择适龄并经鉴定的在Tet-on系统调控下肝脏特异性表达Cre重组酶的双转基因小鼠Lap/LC-1与在Tet-on系统调控下肝脏特异性表达萤光素酶(Luc)的双转基因小鼠Lap/NS3/4A交配,子代小鼠经PCR检测、筛选基因组中NS3/4A、Lap、LC-1等3个转基因片段均阳性的小鼠。三阳性的NS3/4A/Lap/LC-1小鼠经多西环素(Dox)诱导1周后,以在体生物发光成像系统(BLI)检测报告基因Luc的表达,免疫组化检测小鼠体内Cre重组酶、HCV NS3/4A丝氨酸蛋白酶的表达状况。结果:NS3/4A/Lap/LC-1小鼠经Dox诱导后,BLI结果显示仅在小鼠肝脏部位有强烈的发光信号,表明这些小鼠肝细胞内报告基因Luc特异高效表达;免疫组化结果证实Cre重组酶、NS3/4A蛋白酶仅在经诱导后的小鼠肝细胞中特异性表达。结论:建立了Tet-On调控系统和Cre/loxP基因剔除系统双重调控下表达HCV NS3/4A丝氨酸蛋白酶的三转基因小鼠模型,为进一步研究HCV NS3/4A丝氨酸蛋白酶在HCV感染后与宿主相互作用的机制,以及抗NS3/4A丝氨酸蛋白酶特异性抑制剂的筛选奠定了基础。     

Syncytin-A mediates the formation of syncytiotrophoblast involved in mouse placental development.

Syncytin-A, a new mouse endogenous retroviral envelope protein expressed in placenta, can mediate cell fusion in vitro. But its physiological function was still unknown. We proposed a role for syncytin-A in syncytiotrophoblast (SynT) formation derived from the differentiation of trophoblast stem (TS) cells during placental development. To evaluate this hypothesis, we analyzed the involvement of syncytin-A in the differentiation of mouse TS cells. After withdrawing fibroblast growth factor 4 (FGF4), TS cells can fuse to form SynT cells. We found syncytin-A mRNA and protein expression are colinear with fusion index increase during TS cell differentiation. Expression of syncytin-A is localized in SynT cells through in situ immunofluorescent staining. By using specific antibody and antisense oligonucleotides, we demonstrated that inhibition of syncytin-A lead to obvious decrease of SynT cell formation. These results present evidence in support of the direct role for syncytin-A in mouse TS cell fusion and differentiation involved in placental development.     

Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.     

Non‐parallel recombination limits cre‐loxP‐based reporters as precise indicators of conditional genetic manipulation

Cre/LoxP‐mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage‐tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre‐expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre‐mediated recombination, which raises concerns regarding utilization of Cre‐reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre‐expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non‐parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent‐target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations. genesis 51:436–442. © 2013 Wiley Periodicals, Inc.     

Generation and characterization of PDGFRα‐GFPCreERt2 knock‐In mouse line

Platelet‐derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα‐expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα‐expressing cells in vivo, we generated a knock‐in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand‐binding domain (ER^T2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα‐expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock‐in mouse line generated here could be useful for studying PDGFRα‐expressing cells and their descendants in vivo at various stages of development. genesis 53:329–336, 2015. © 2015 Wiley Periodicals, Inc.