Germ line transformation of mammals by pronuclear microinjection

Rülicke, T.; Hübscher, U.

doi:10.1017/s0958067000020923

Cited by 14 publications

(11 citation statements)

References 40 publications

(45 reference statements)

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“…The experimental introduction of DNA (transgenes) into the germ line provides an opportunity to probe an organism’s response to foreign DNA (Rulicke and Hubscher, 2000), and has revealed that organisms use a variety of mechanisms to silence transgenes in the germ line (Birchler et al, 2003; Brodersen and Voinnet, 2006). Interestingly, some mutants that disrupt transgene silencing also de-silence endogenous genes, including self-replicating elements called transposons (Ketting et al, 1999; Tabara et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline

Shirayama

Seth

Lee

et al. 2012

Cell

566

875

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SUMMARY Organisms exhibit a fascinating array of gene-silencing pathways, which have evolved in part, to confront invasive nucleic acids such as transposons and viruses. A key question raised by the existence of these pathways is how do they distinguish “self” from “non-self” nucleic acids? Evidence exists for a number of mechanisms that might facilitate detection of foreign sequences including mechanisms that sense copy-number, unpaired DNA, or aberrant RNA (e.g. dsRNA). Here we describe an RNA-induced epigenetic silencing pathway, RNAe, that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate RNAe, while maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences, while two other Argonaute pathways serve as epigenetic memories of “self” and “non-self” RNAs. These findings suggest how organisms may utilize RNAi-related mechanisms not only to recognize and silence foreign genes, but also to keep inventory of all genes expressed in the germ-line.

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Section: Introductionmentioning

confidence: 99%

piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline

Shirayama

Seth

Lee

et al. 2012

Cell

566

875

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“…The percentage of efficiency for getting a transgene positive animal was found to be about 46% by this method, which was higher than the conventional method of transgensis by pronuclear DNA injection method 10–20%, 4,5 but lower than the earlier method which used gene electroporation in the testis. 7 Although we found the overall transgenic efficiency for this method is 46% (positive animals from all constructs taken together), Bucsn2-IRES2-Egfp and Fetuin A-shRNA the efficiency (70%) was found to be higher (70%) and lower (32%), respectively than the average.…”

Section: Discussionmentioning

confidence: 56%

“…For instance, classical method of gene transfer involves microinjection of nucleic acids into fertilized eggs, typically yielding low success rate of ~10–20%. 4,5 Moreover, this technique is beyond reach of common researcher, largely due to technical complexities in adopting skills for micro manipulation of embryos. 4 This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research.…”

Section: Introductionmentioning

confidence: 99%

Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells

Usmani

Ganguli

Jain

et al. 2016

Molecular Therapy - Methods & Clinical Development

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Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

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“…The use of expression cassettes in mammals suffers from the difficulty of identifying key regulatory elements, such as enhancers or silencers, that are necessary for the correct expression of a transgene [6]. A related source of variability is that expression of the label is influenced by the DNA sequences that flank the inserted DNA, yet the site of integration into the genome differs between transgenic animals.…”

Section: Labeling Of Specific Neuronal Phenotypes—mus Musculus As a “mentioning

confidence: 99%

From Art to Engineering? The Rise of In Vivo Mammalian Electrophysiology via Genetically Targeted Labeling and Nonlinear Imaging

Kleinfeld¹,

Griesbeck²

2005

PLoS Biol

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Germ line transformation of mammals by pronuclear microinjection

Abstract: 591Figure 2Development of pronuclei and microinjection of DNA into the mouse zygote. A and C, development of pronuclei (arrows), about 20 and 26 h post hCG-injection for superovulation; B, DNA-microinjection into the male pronucleus; D, pronuclei disappear before the first cell division.

Cited by 14 publications

References 40 publications

piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline

piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline

Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells

From Art to Engineering? The Rise of In Vivo Mammalian Electrophysiology via Genetically Targeted Labeling and Nonlinear Imaging

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