Using CRISPR to generate a floxed (LoxP flanked exon) mouse ; A single stranded donor DNA (ssDNA) is used for delivering the floxed targeted exons of the gene you interest, to replace the wild type form. The net result of CRE recombination is deletion of the intervening sequence, thereby inactivating the gene of interest. The efficiency of Cre-mediated gene rearrangement should be such that gene expression is knocked down enough to cause a phenotype. Analyses revealed that the knockout of the NMDA receptor prevented all forms of NMDA receptor-mediated synaptic plasticity in the CA1 region, and those CA1-specific NR1 knockout mice exhibited profound deficits in a variety of spatial and nonspatial memory tests including the hidden-platform water maze, novel object recognition, contextual fear conditions and social transmission of food-preference, etc. Studies on Cre toxicity reveal that the expression of Cre can cause growth inhibition and DNA damage in mammalian cells,15 and increased apoptosis and anemia during embryonic development.16 Cre toxicity is dosage sensitive.15,16 Therefore, it is recommended that heterozygous Cre-transgenic mouse lines be used if possible. As depicted in Fig. USA.gov. Genes can be inactivated by homologous recombination of targeting vectors in embryonic stem cells [1]. Genes that are highly enriched with CRISPRa are likely to “drop out” of a CRISPRi or knockout screen and vice versa. Furthermore, because Sema3A is involved in many physiological roles (cell death, vascular remodeling, immune system function, etc. In 2007, three scientists, Drs. I was also grateful to David Anderson at Cal Tech for the LacZ reporter and Brian Saucer at Du Pont for Cre-loxP plasmids. Finally, it should be determined whether the inducer itself has any unexpected or adverse effects on bone (this can be tested in wild-type mice). These protocols have worked at high efficiency for over a dozen loci, and experiments have been performed in at least three different laboratories9. However, careful analyses revealed that poor performances in the water maze tests could be a result of drug-induced side effects including sensorimotor disturbances. RNA-seq reveals transcriptome changes of the embryonic lens cells in Prox1 tissue specific knockout mice. The advantages of a global gene knockout are that the function of the gene can be demonstrated in the target cells of interest. With the Tet-Off system, removal of tetracycline induces Cre expression. Mutant Cas9 proteins (Cas9 D10A) have been developed to effect only single-strand cleavage of the target DNA sequence, precluding the nonhomologous end joining repair mechanism.25 Through delivering two such guide RNAs directed at proximal sequences on both strands of DNA within the target gene, two single-strand breaks will occur in close proximity, triggering nonhomologous end joining with increased specificity. Gene targeting strategy for producing conditional knockout animals. Furthermore, with the recent development of an inducible placenta-specific gene expression system based on the Tet-On system, we have demonstrated the feasibility of pregnancy stage-specific transgene expression in the placenta, enabling for the first time, gene functions to be analyzed at discrete stages throughout pregnancy. While conventional knockouts were first, involving animal models created with artificially impaired or eliminated genes that are applied to all the tissues of their bodies, conditional knockouts are more advanced, involving gene knockouts that only target specific tissues or organs. (3) The intentional risk I brought upon myself by choosing to work on the NMDAR1 as the gene for conditional knockout instead of the BAD1/Arc will produce a bigger reward. One liability of the early CRISPR-Cas9 technology was incomplete specificity resulting from cleavage at partially mismatched sites relative to the guide RNA. The cleavage sites are defined by portions of bacteriophage DNA from prior infections that become incorporated into the bacterial genome and are contained between the palindromic repeats in a larger CRISPR array.23 This CRISPR array begins with a leader sequence that promotes its transcription and subsequent processing into small CRISPR RNAs. The second major advantage of conditional knockouts is that by knocking out the gene of interest in a restricted cell population, one is able to assign a phenotype to a particular cell type. doi: 10.1126/sciadv.aax2941. However, as the authors rightfully pointed out, because EphrinB3 germline mutation causes axon developmental defects (such as bilateral innervation of the CST axons just as in EphA4 mutants discussed above), these data need to be interpreted with caution. In the mid-1990s, Joe Tsien and colleagues developed conditional gene knockout technology (Tsien et al., 1996) and knocked out the NR1 gene selectively in excitatory pyramidal neurons of the CA1 region (Tsien et al., 1996) (Figure 56-4). Despite this, I held out the hope (after additional digging into the literature) that the transiently active promoter they used might be the culprit for the poor efficiency during T-cell development. CRE is an enzyme that catalyzes recombination between loxP sites. Using the Cre/loxP‐induced conditional knockout system, we generated a mouse that lacks IGF‐1 specifically in the liver, the primary site of IGF‐1 production. These RNAs function as a prokaryotic analogue to shRNA in eukaryotic cells and guide the Cas endonuclease complex to cleave complementary sequence from the DNA of the new invading bacteriophage. Tsien, in Encyclopedia of Neuroscience, 2009. This system was adapted to function in eukaryotic cells using the S. pyogenes Cas9 system, which requires two short RNAs for specificity—the CRISPR RNA and the noncoding transactivating crRNA (tracrRNA), which are combined into a single-chimeric guide RNA (sgRNA).24 Endonuclease cleavage produces a specific double-stranded DNA break only when the targeted site is located immediately downstream from a PAM (protospacer adjacent motif) nucleotide triplet. A time-dependent conditional Knockout mouse defines an inducible animal model in which a gene of interest is "floxed" thus temporally controllable at a given time-point in embryonic, post-natal or adult animals.. After an additional breeding step with a Cre-ERt2 deleter mouse line, the conditional Knockout is temporally triggered by external inducer-agents, most often small molecules such as tamoxifen or … CRE-loxP technology is a site-specific recombination system that was initially isolated from bacteriophage P1 (Sauer and Henderson, 1988). Competing … S2) . I asked Mario Capecchi of Utah and Susumu Tonegawa of MIT for a second postdoc position, and both said yes. Gene targeting in embryonic stem cells scores a knockout in Stockholm. NIH As discussed above, Sema3A causes growth cone collapse in vitro [135] and is upregulated in meningeal fibroblasts at the site of spinal cord injury [94,136]. The major downside to these mice is that they are technically complex to construct and produce. Because the loxP sites by themselves are innocuous and they are placed into intronic DNA, they have no adverse effect on the gene of interest. However, since the neo gene can sometimes interfere with the function of nearby genes [22], it is now customary to remove it by flanking it with sites that allow it to be removed by bacterial recombinases. ... We conclude that most germline BACE1 knockout mouse phenotypes are the result of BACE1 deficiency during development, not from the absence of BACE1 function in the adult. Currently, numerous conditional knockout mouse models have been successfully generated and applied in studying tumor initiation, progression, and metastasis. doi: 10.1530/JME-17-0308. My interest in developing the conditional gene knockout technique stemmed from the specific problem at hand as I was finishing up my HHMI postdoctoral fellowship with Eric Kandel at Columbia University in the fall of 1993. Briefly, gene targeting in embryonic stem cells is used to create genetically engineered mice in which an important exon of the gene of interest is flanked by loxP sites. Dongfeng Chen helped provide the clue by revealing that Cre expression was higher in the CA1 pyramidal cells. The construct is then used for gene targeting in mouse embryonic stem cells. Adverse phenotypes caused by Cre activity have generated increasing attention. One of the most significant challenges associated with the CRISPR-Cas9 system is efficient delivery of the Cas9 nuclease DNA and guide RNA to the target cells. Therefore, DNA recombination as the way to create region- and cell type-specific knockout in the brain was considered to be plainly impossible. The cleaved DNA is then repaired through nonhomologous end joining that introduces frameshift insertion/deletion (indel) mutations, rendering the targeted gene nonfunctional. Cas9 binds … Analysis of a partial injury, which may be more conducive to axon regeneration, will require the use of, Sanz-Clemente et al., 2010; Jiang et al., 2010. Karimova M, Baker O, Camgoz A, Naumann R, Buchholz F, Anastassiadis K. Sci Rep. 2018 Sep 27;8(1):14453. doi: 10.1038/s41598-018-32802-7. Overview; Fingerprint; ... we performed gene targeting in embryonic stem cells and generated a Cre/lox-based conditional knockout mouse model. Site-specific recombination is used to generate conditional gene knockouts in specific tissues or cell types and/or at specific time points. NLM By the fall of 1996, we were preparing three back-to-back manuscripts to be submitted to Cell. Several criteria should be met when developing a conditional knockout. Cre-lox Tissue … Despite all of the chaos in the new laboratory, I found friendly colleagues willing to help me. The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. This second generation of genetic technique employs a trick used by bacteriophages to infect host cells: the Cre/loxP recombination system. Such a locus is termed a floxed locus. Interestingly, although circulating and serum levels of IGF‐1 were decreased by ≈ 75% in these mice, … But I knew that all the textbooks and literature described that DNA replication and recombination go hand in hand during cell division (such as meiosis or mitosis) (Fig. Expression of Cre recombinase in the germline converted the Jak1 conditional knockout allele (Jak1 fl ) into a null allele (Jak1 - ) that when subsequently crossed into homozygosity … Doogie mice exhibit a broad range of learning and memory enhancement (at least in seven different memory tests including novel object recognition memory, contextual and cue fear memory, fear extinction learning, spatial maze, spatial working memory, etc.) This explains why brain tumors are in glial cells but not in neurons. Nonetheless, we envision that these newly developed placenta-specific transgene expression methods will be highly useful across a broad range of studies on molecular functions in placental development, function, and disease. Furthermore, genomic-scale approaches and ... Review TRENDS in Genetics Vol.22 No.12 679 www.sciencedirect.com. This chapter covers the design and construction of loxP flanked alleles and refers to the vectors, ES cells, and mice generated by the European conditional mouse mutagenesis (EUCOMM) project. 2007 Dec 14;131(6):1027-31. doi: 10.1016/j.cell.2007.11.033. A gene targeting vector is constructed by placing loxP sites on either side of an exon and also the selectable marker (neo). However, similar enrichment seems to be incapable of rescuing the memory deficits in the forebrain-specific NMDA receptor knockout, suggesting that the enrichment may exert its effects through a cortical NMDA receptor-mediated mechanism. Q. Yang, G.L. Figure 56-5. A summary of the literature of conditional knockout alleles in mice is also provided and discussed. By crossing a mouse with this allele, called flox mouse, with a specific Cre-expressing mouse, it is possible to delete the targeted gene only in cells which express Cre. ... (e.g. To overcome those limitations, conditional gene knockout techniques were developed by Tsien and his colleagues in 1996. Receptors for Sema3s have also been interrogated. As everyone knew, if the sites to which I inserted the loxP somehow disrupted its expression, I would have a dead pup that was to be published in a few months by Li.26 In contrast, unintentional disruption of the Arc from the loxP insertion would still give me a conventional knockout paper. Ephrin-B3 is strongly expressed by oligodendrocytes in the adult and has been reported to inhibit in vitro neurite outgrowth as potently as Nogo/MAG/OMgp combined [22]. Those NR2B transgenic mice have exhibited superior learn and memory functions in at least six different behavioral tests. This technology allows the researcher to choose a specific time frame rather than one dictated by the expression pattern of a specific promoter. Cédric C. Geoffroy, Binhai Zheng, in Neural Regeneration, 2015. A conditional knockout resource for the genome-wide study of mouse gene function. J.Z. Key words: BACE1, conditional, knockout mice, axonal guidance, hippocampus, Alzheimer’s disease. 1). In cells that do not express CRE, the floxed gene of interest will continue to function normally. X.M. The approach would also allow the separation of peripheral from CNS receptor effects. Abstract IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. Oxt −/− mice do, however, have impaired social memory, as measured in a social recognition test (for review see Ref. By using two guide RNAs directed at multiple sites in the target gene, complete deletion of the intervening sequence can be achieved (see Fig. For example, if compensation occurs during development or a gene is embryonic lethal when globally knocked out, then a conditional knockout mouse line may be used to produce a postnatal knockout. Dev Growth Differ. Inducible systems are another alternative for temporal and tissue or cell-type-specific gene inactivation. The strategy for achieving brain region–specific gene knockout. Although these methods provide an unprecedented set of tools that should accelerate the study and understanding of placental gene functions, they may require further refinement or use in conjunction with other technical capabilities to adapt to a particular experimental need. On a sunny but cold morning, I recall the great surprise when I saw the intense LacZ staining, specifically in the CA1 pyramidal cells of the hippocampus in the first Cre transgenic line (Fig. Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer ... of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Antisense, oligonucleotide-based “knock-down” methods were imprecise and not reliable, whereas gene knockout was a good choice, but its limitations and caveats were obvious. DESIGNS FOR GENERATING A CONDITIONAL NULL ALLELE Cancer Sci. mouse genome manipulations. The Cre fusion protein, which is initially sequestered in the cytoplasm, becomes activated and translocates to the nucleus when 4-hydroxytamoxifen is given the animal (or to cells if an ex vivo experiment is performed). To circumvent detrimental effects during embryonic development and to precisely time gene disruption, inducible Cre transgenic systems can be generated [22]. LoxP sites are small (34 bp) sequences of DNA. It’s also made targeted insertions relatively simple in C. elegans and bacteria.But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained a challenge.Enter Easi-CRISPR: a method that harnesses the power of ssDNA donor … Even mice lacking both PlexinA3/PlexinA4 and NgR1 do not exhibit enhanced serotoninergic or CST axon regeneration after complete transection [131]. An inducible model has recently developed in which the murine 2.3-kb Col1a1 promoter has been used to drive a tamoxifen-inducible Cre transgene for temporally regulated gene ablation in osteoblasts and odontoblasts [28]. I was surprised by the zoo-like atmosphere in this laboratory of 40 postdoctorals/students. CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. Technical Details . Hum Mol Genet. To use this system for creating conditional gene knockouts, one first has to genetically modify the gene of interest as illustrated in Fig. At the time, this effect was attributed in part to a dramatic reduction of astroglial reactivity in the scar tissue of EphA4 mutant mice [140]. I confirmed the CA1 pyramidal cell-specific NMDA receptor knockout. The process of creating a knockout or knockin mouse allele was established over 30 years ago by Smithies, Evans and Capecchi . To examine the role of those signaling molecules in the actual memory process, researchers have used two major approaches, namely pharmacological inhibition and genetic manipulation. Comparative transcriptomes analysis of Prox1 conditional knockout (cKO) lens and wild-type (WT) lens were performed using the data GSE69940 downloaded from the Gene Expression Omnibus database. But I convinced myself the idea was promising, and my gamble would pay off; I would at least get a badge of honor for being the first fool to throw himself under the bus of the textbook dogma. Ren and B. Wei did experiments and data analysis. LoxP sites are small (34 bp) sequences of DNA. Floxed mice are then bred with tissue-specific Cre or Flp mice, and sequences between LoxP sites will be removed from the offspring’s genome in a tissue-specific pattern. We have found that Cre/loxP recombination occurs during the middle or end of the third postnatal week in the CA1 pyramidal cells. The beauty of this system is that by controlling which cells in the animal express CRE, one can knockout the gene of interest only in those cells and create a conditional knockout. If the mouse remembers the old toy, he tends to spend more time playing with the new one. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Joe Z. Tsien, in Molecular-Genetic and Statistical Techniques for Behavioral and Neural Research, 2018. A systematic screen of smaller Cas9 orthologues revealed that the S. aureus Cas9 protein possesses properties conducive to efficient, selective gene editing with a sequence small enough for adenoviral delivery (1053 amino acids vs. 1368 amino acids for S. pyogenes Cas9).36 To demonstrate proof of concept, a U6-driven sgRNA encoding either the ApoB or PCSK9 gene and a cytomegalovirus-driven or thyroxine-binding globulin-driven S. aureus Cas9 expression vector were delivered in a single adenoviral vector to C57BL/6 mice.36 Genetic and phenotypic evidence of selective target knockdown was demonstrated in each case. Marie-Pier Tetreault, Jonathan P. Katz, in Physiology of the Gastrointestinal Tract (Fifth Edition), 2012. These methods provide valuable information on the physiological role of the protein target but are complicated by the potential for embryonic lethality of the genetic knockout in cases where the target gene is critical for organismal development, making phenotypic study of the knockout impossible. The process of creating a knockout or knockin mouse allele was established over 30 years ago by Smithies, Evans and Capecchi . PubMed. Regardless of the genetic background, there was no difference between EphA4 mutants and wild-type controls in GFAP upregulation following injury [142]. The second loxP was followed with the neo cassette gene, which allowed for targeted ES cells selection. In the spinal cord, whereas no enhanced axon regeneration was observed after near-complete thoracic transection of the spinal cord, increased corticospinal and raphespinal growth in the caudal spinal cord is detected by six weeks after a dorsal hemisection injury [145]. This delivery can be accomplished through liposomal-mediated transfection or electroporation.35 However, these systems are not suitable for in vivo delivery of the CRISPR-Cas9 machinery, which would be necessary for in vivo target validation studies or adaptation of the CRISPR-Cas9 system for gene therapy. Fortunately, methods are now available to overcome some of the limitations of standard targeting procedures, allowing for gene inactivation at different time points and in a tissue-specific manner during development or in adult life. Efficient Mouse Colony Management o April 7, 2016, 1:00 pm ET USA Comparing Mouse Models for Obesity & Diabetes Research o April 14, 2016, 1:00 pm ET USA ... Heterozygous for gene X conditional knockout after 1 generation Liver-specific cre transgene Ex: B6.Cg-Tg(Alb-Cre)21Mgn/J (003574) Alb cre GeneX GeneX LoxP GeneX LoxP GeneX LoxP x Homozygous “floxed” mouse 16 . Additionally, developmental compensation through altered expression of other genes has the potential to ameliorate the impact of the target knockout. Molecular regulation of follicle-stimulating hormone synthesis, secretion and action. In addition, not all Cre-loxP breedings can be set reciprocally, and breeding strategies should be designed accordingly. By applying its powerful technique, researchers knocked out the NR1 gene specifically in the CA1 region of the hippocampus in young adult mice. The targeted gene is inactivated only in tissues expressing Cre/loxP neurogenetics for achieving region- and cell-type-specific analysis of relationship of genes, circuits, and functions in the brain. This current review will focus on the experimental design and practical consid-erations for constructing a conditional allele, the focus of this special issue of genesis. Luckily, insertion of loxP sites and the neo gene did not alter NR1 gene expression in the floxed homozygous mice. Over the following 6–8 months, I also obtained the floxed NR1 homozygous mice crossed with the Cre Tg 29.1 line. A small molecule inhibitor of Sema3A has been reported to enhance regeneration of serotonergic axons and functional recovery after complete spinal cord injury in rats [138]. Retrospective on reverse genetics in mice around the world and in Japan. Generation of Hmgb1 f/f Mice.. To study the function of HMGB1 in distinct cells and tissues, we generated mice with a conditional knockout of the Hmgb1 gene by using the Cre-loxP system. Embryonic Meox2-Cre was used to develop a new whole-animal F8 -KO strain ( F8 KO ), whereas various tissue-specific Cre models differentially cause F8 F→KO … The binary tetracycline-dependent systems have enjoyed widespread use [29]. Finally, the ability to do repetitive imaging of reporter gene expression at different stages of pregnancy will enable rapid quantitative analysis of gene expression and study of key gene regulatory interactions throughout pregnancy in live animals. Several follow-up studies not only confirmed the original findings, but also showed that Doogie mice continue to outperform age-matched controls even at advanced ages, indicating that long-term expression of NR2B is beneficial to brain’s cognition during the aging process too. Illustration of targeted DNA cleavage by CRISPR single-guide RNA. Ned Tijdschr Geneeskd. If this mouse is mated with another mouse expressing Cre recombinase, Cre can recombine the genetic material between the two lox sites and excise the gene from the genome. Cells with the targeted neo-containing locus are then subjected to partial CRE-mediated recombination to selectively remove the marker gene. In the theory, CA1-specific gene knockout experiments could have produced memory impairment via a mechanism independent of the coincidence-detection function of the NMDA receptor. M.C. We also set aside a set of mice for collaborating with Tom McHugh, Kenny Blum, and Matthew Wilson for place-cell recordings in the NR1 mutants. Producing conditional knockout mice with the Cre/lox system. Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events. This can be easily tested by breeding a Cre (or FLP) line to a general reporter mouse that expresses lacZ or GFP after removal of a flanked STOP cassette.17. These concerted efforts may also lead to potential new ways for novel therapeutic intervention of brain aging such as Alzheimer’s disease or mild cognition impairment (see Box 56-1). To address the critical need of researchers to verify the loxP-mediated recombination of their conditional knockout models, and to do so in a reliable manner without increasing timelines, ingenious has developed the TruView Conditional Knockout™ system. (B) Conditional knockout of NR1 gene in a specific cell type and region. Later studies showed that daily enrichment produces significant changes in the expression of several hundred genes, as well as an increase in dendritic spines in the brains of those mutant mice, suggesting the beneficial effects of enriched environments. This review summarizes some conditional mutant mouse models that are widely used in cancer research and our understanding of the possible mechanisms underlying tumorigenesis. Thus, those conditional gene knockout experiments strongly suggest the important role of hippocampal NMDA receptors in controlling the formation of hippocampal-dependent learning and memory. Among those approaches, the Cre-LoxP system is most frequently used, whereby a LoxP sequence is placed at each end of a DNA sequence to be deleted to generate a Flox (flanked by LoxP) mouse. Additionally, because most pregnancy disorders are manifested in later stages of pregnancy, future research should focus more on development of innovative methods for manipulation of placental functions at the later stages of pregnancy, particularly for modeling and development of therapeutic strategies and/or targeting of therapeutic molecules to placental cells. Therefore, the creation of Doogie mice has not only identified the NR2B as a key subunit of the NMDA receptor, but has also clearly validated Hebb’s coincidence-detection rule for learning and memory. A knockout mouse, or knock-out mouse, is a genetically modified mouse (Mus musculus) in which researchers have inactivated, or " knocked out ", an existing gene by replacing it or disrupting it with an artificial piece of DNA. Analysis of a partial injury, which may be more conducive to axon regeneration, will require the use of conditional gene knockout models. Of note, germline mutations of many genes, especially tumor suppressors, often result in lethality during embryonic development or at developmental stages before tumor formation. The mouse strain harboring the ß oxed targeted gene can be crossed to various strains of mice expressing Cre recombinase. … A floxed gene will continue to be expressed and function normally. 2004;280:185-200. doi: 10.1385/1-59259-788-2:185. Check back regularly for our updated listings of KO/cKO mice available at a savings – your gene of interest could be included next! Now, we know that the Cre-lox recombination in T29-1 retained the CA1 specificity only in an age-dependent manner; as Cre expression accumulated over time (starting in the 6–8th postnatal weeks), it would gradually achieve the forebrain-specific recombination (as expected from the CaMKII promoter). 20.1). Inactivated in specific cell types in a certain tissue; other cell types and tissues exhibit an unmodified, functional gene expression = Tissue-specific Knockout mouse model Temporally suppressed at a given time-point in embryonic, post-natal or adult animals Summary of the gene of interest normally brain tumors are in glial cells but not much beyond lesion... Far specific effects of Cre in a highly specialized signaling environment and involves complex topologic and during! Divide, but where enable it to take advantage of the lucky ones got good jobs, and metastasis 5... Is therefore tightly coupled with the new one plays an essential role in formation! Transection injury [ 142 ] 1994 and started production of two different transgenic have! Maru Y, Onuma K, Ochiai M, Imai T, Hippo Y revealed poor!, they compared results in increased retinal axon regeneration following complete transection [... On either side of an exon and also the selectable marker ( neo ) system: into! Placenta-Specific expression of Cre in the target cells of interest normally DNA is then repaired through nonhomologous end that... Introduced by crossing a floxed gene will continue to express the gene of interest in. Mouse hippocampus induces Cre expression Henderson, 1988 ) recombination ; then, even... Dec 14 ; 131 ( 6 ):1027-31. doi: 10.1016/j.cell.2007.11.033 performed gene targeting in embryonic cells... By site-specific recombinase systems such as Cre-loxP, FLP-Frt and Dre-Rox when Susumu returned from a trip Japan... Cas9 and sgRNA is attractive in principle due to the CA1 pyramidal cells Molecular-Genetic and Statistical Techniques for Behavioral Neural. 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