One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

CRISPR/Cas systems are widely used to knock out genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no ‘two-in-one’ KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.

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Decision letter: One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

10.7554/elife.48081.021 ◽

2019 ◽

Author(s):

David Grunwald

Keyword(s):

Conditional Knockout ◽

Dual Function ◽

Efficient Generation ◽

One Step

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Author response: One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

10.7554/elife.48081.022 ◽

2019 ◽

Cited By ~ 1

Author(s):

Wenyuan Li ◽

Yage Zhang ◽

Bingzhou Han ◽

Lianyan Li ◽

Muhang Li ◽

...

Keyword(s):

Conditional Knockout ◽

Author Response ◽

Dual Function ◽

Efficient Generation ◽

One Step

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One-step Generation of Zebrafish Carrying a Conditional Knockout-Knockin Visible Switch via CRISPR/Cas9-Mediated Intron Targeting

10.1101/827352 ◽

2019 ◽

Author(s):

Jia Li ◽

Hong-yu Li ◽

Shan-ye Gu ◽

Hua-Xing Zi ◽

Lai Jiang ◽

...

Keyword(s):

Animal Model ◽

Homologous Recombination ◽

Gene Knockout ◽

Conditional Knockout ◽

Egfp Expression ◽

Fluorescent Reporters ◽

One Step ◽

Step Generation ◽

Conditional Gene Knockout ◽

Low Efficiency

SUMMARYThe zebrafish has been becoming a popular vertebrate animal model in biomedical research. However, it is still challenging for making conditional gene knockout (CKO) zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zCKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-inv associated with TagRFP expression, enabling to visualize CKO alleles. Thus, the simplification of the design, and the visibility and combination of both CKO and KI alleles’ engineering make our zCKOIS strategy an applicable CKO approach for zebrafish.

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Clinical and microbiological aspects of vaginal microbiota at pregnant with threat of late miscarriages during treatment with vaginal gestagens forms

HEALTH OF WOMAN ◽

10.15574/hw.2017.117.68 ◽

2017 ◽

pp. 68-73

Author(s):

I.P. Polishchuk ◽

Keyword(s):

Pregnant Women ◽

Therapeutic Effect ◽

Local Treatment ◽

Vaginal Microbiota ◽

Control Group ◽

Before And After ◽

Low Efficiency ◽

Late Miscarriage ◽

Microbiological Aspects ◽

Micronized Progesterone

The objective: was to examine the effectiveness of treatment of late miscarriage threat by micronized form of progesterone for 100 mg – 3 times a day in the form of gelatin pills and vaginal tablets with lactose. Patients and methods. Under our supervision there were 70 pregnant women with normocenosis of vagina (NCV) without extragenital pathology, which were not performed systemic or local treatment with antibacterial drugs in the last 4 weeks. Among them 25 pregnant women with TLSM treated by gelatin tablets of micronized progesterone (GTP) (group 1); 25 pregnant women with TLSM, treated by vaginal micronized progesterone tablets (VPT) (2nd group) and 20 healthy women with physiological pregnancy – PV (control group). The distribution of women in the group adhered to the principles of randomization. The age of examined women ranged from 19 to 32 years, most pregnant women were aged under 30 years (89.02%). General clinical examination was carried out according to the standard scheme according to the Order MH of Ukraine № 620. Results. During the research we have determined the colpocytologcal dynamics and state of vaginal microbiota in pregnant women with threatened late miscarriage with initial vaginal normocenosis before and after treatment whit vaginal forms of progesterone. Conclusion. The received results showed low efficiency of micronized progesterone gelatin dragee at threat of the late miscarriage that at small therapeutic effect has led to the development of vaginal dysbiosis in all surveyed. In contrast, the use of micronized progesterone vaginal tablets – the maximally rapid therapeutic effect without disturbance of vaginal normocenosis. Key words: the threat of a late miscarriage, vaginal micronized forms of progesterone.

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Pharmaceutical and Biotechnological Applications of Transgenic Technology

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2018.11.4.1 ◽

2018 ◽

Vol 11 (4) ◽

pp. 4145-4153

Author(s):

D Samba Reddy ◽

Tina Reddy

Keyword(s):

Mammalian Species ◽

Genetic Diseases ◽

Transgenic Animals ◽

Transgenic Animal ◽

Pharmaceutical Products ◽

Conditional Knockout ◽

Knock Out ◽

Transgenic Lines ◽

Health And Disease ◽

Or Genes

A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined. They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth). The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine. Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products. In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.

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Single Step 2-Port Device De-Embedding Algorithm for Fixture-DUT-Fixture Network Assembly

Electronics ◽

10.3390/electronics10111275 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1275

Author(s):

Simone Scafati ◽

Enza Pellegrino ◽

Francesco de Paulis ◽

Carlo Olivieri ◽

James Drewniak ◽

...

Keyword(s):

Standard Technique ◽

Single Step ◽

Scattering Parameters ◽

Linear Set ◽

S Parameter ◽

Parameter Conversion ◽

Before And After ◽

One Step ◽

Typical Measurement

The de-embedding of measurement fixtures is relevant for an accurate experimental characterization of radio frequency and digital electronic devices. The standard technique consists in removing the effects of the measurement fixtures by the calculation of the transfer scattering parameters (T-parameters) from the available measured (or simulated) global scattering parameters (S-parameters). The standard de-embedding is achieved by a multiple steps process, involving the S-to-T and subsequent T-to-S parameter conversion. In a typical measurement setup, two fixtures are usually placed before and after the device under test (DUT) allowing the connection of the device to the calibrated vector network analyzer coaxial ports. An alternative method is proposed in this paper: it is based on the newly developed multi-network cascading algorithm. The matrices involved in the fixture-DUT-fixture cascading gives rise to a non-linear set of equations that is in one step analytically solved in closed form, obtaining a unique solution. The method is shown to be effective and at least as accurate as the standard multi-step de-embedding one.

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One-step synthesis and performances of Ni@CN nanocapsules with superior dual-function as electrocatalyst and microwave absorbent

Colloids and Surfaces A Physicochemical and Engineering Aspects ◽

10.1016/j.colsurfa.2021.126162 ◽

2021 ◽

pp. 126162

Author(s):

Shu-Tong Li ◽

Gui-Mei Shi ◽

Qian Li ◽

Fa-Nian Shi ◽

Xiao-Lei Wang ◽

...

Keyword(s):

Dual Function ◽

One Step

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Durability of Lubricated Icephobic Coatings under Multiple Icing/Deicing Cycles

10.31399/asm.cp.itsc2021p0473 ◽

2021 ◽

Author(s):

Valentina Donadei ◽

Heli Koivuluoto ◽

Essi Sarlin ◽

Petri Vuoristo

Keyword(s):

Industrial Applications ◽

Power Lines ◽

Operational Performance ◽

Flame Spraying ◽

Mechanical Interlocking ◽

Properties Of Coatings ◽

Before And After ◽

Outdoor Environments ◽

One Step ◽

Ice Adhesion

Abstract In subzero conditions, atmospheric ice naturally accretes on surfaces in outdoor environments. This accretion can compromise the operational performance of several industrial applications, such as wind turbines, power lines, aviation, and maritime transport. To effectively prevent icing problems, the development of durable icephobic coating solutions is strongly needed. Here, the durability of lubricated icephobic coatings was studied under repeated icing/deicing cycles. Lubricated coatings were produced in one-step by flame spraying with hybrid feedstock injection. The coating icephobicity was investigated by accreting ice from supercooled microdroplets using an icing wind tunnel. The ice adhesion strength was evaluated by a centrifugal ice adhesion tester. The icing performance was investigated over four icing/deicing cycles. Surface properties of coatings, such as morphology, topography, chemical composition and wettability, were analyzed before and after the cycles. The results showed an increase in ice adhesion over the cycles, while a stable icephobic behaviour was retained for one selected coating. Moreover, consecutive ice detachment caused a surface roughness increase. This promotes the formation of mechanical interlocking with ice, thus justifying the increased ice adhesion. Finally, the coating hydrophobicity mainly decreased as a consequence of the damaged surface topography. In summary, lubricated coatings retained a good icephobic level after the cycles, thus demonstrating their potential for icephobic applications.

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