scholarly journals An Efficient CRISPR protocol for generating Conditional and Knock-in mice using long single-stranded DNA donors

2017 ◽  
Author(s):  
Hiromi Miura ◽  
Rolen M. Quadros ◽  
Channabasavaiah B. Gurumurthy ◽  
Masato Ohtsuka
Keyword(s):  
Mouse Models ◽  
Knockout Mouse ◽  
Gene Sequence ◽  
Gene Replacement ◽  
Conditional Knockout ◽  
Efficient Technology ◽  
Exogenous Dna ◽  
Highly Efficient ◽  
Double Stranded Dna ◽  
Or Gene

The CRISPR/Cas9 tool 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. The majority of mouse models used in research are knock-in (reporters or recombinases) or gene-replacement (for example, conditional knockout alleles containing LoxP sites flanked exons). A few methods for creating such models are reported using double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs serve as very efficient donors, both for insertion and for gene replacement. We call this method Easi-CRISPR (efficient additions with ssDNA inserts-CRISPR), a highly efficient technology (typically 25%-50%, and up to 100% in some cases), one that has worked at over a dozen loci thus far. Here, we provide detailed protocols for Easi-CRISPR.

scholarly journals Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors

2017 ◽  
Vol 13 (1) ◽  
pp. 195-215 ◽  
Author(s):  
Hiromi Miura ◽  
Rolen M Quadros ◽  
Channabasavaiah B Gurumurthy ◽  
Masato Ohtsuka
Keyword(s):  
Mouse Models ◽  
Knockout Mouse ◽  
Conditional Knockout ◽  
Conditional Knockout Mouse

scholarly journals Does murine spermatogenesis require WNT signalling? A lesson from Gpr177 conditional knockout mouse models

2016 ◽  
Vol 7 (6) ◽  
pp. e2281-e2281 ◽  
Author(s):  
Su-Ren Chen ◽  
J-X Tang ◽  
J-M Cheng ◽  
X-X Hao ◽  
Y-Q Wang ◽  
...  
Keyword(s):  
Mouse Models ◽  
Knockout Mouse ◽  
Wnt Signalling ◽  
Conditional Knockout ◽  
Conditional Knockout Mouse

scholarly journals Granulosa Cell-Specific Inactivation of Follistatin Causes Female Fertility Defects

2004 ◽  
Vol 18 (4) ◽  
pp. 953-967 ◽  
Author(s):  
Carolina J. Jorgez ◽  
Michal Klysik ◽  
Soazik P. Jamin ◽  
Richard R. Behringer ◽  
Martin M. Matzuk
Keyword(s):  
Transgenic Mice ◽  
Granulosa Cell ◽  
Mouse Models ◽  
Premature Ovarian Failure ◽  
Knockout Mouse ◽  
Severe Case ◽  
Ovarian Failure ◽  
Conditional Knockout ◽  
Specific Deletion ◽  
Female Physiology

Abstract Follistatin plays an important role in female physiology by regulating FSH levels through blocking activin actions. Failure to regulate FSH has been implicated as a potential cause of premature ovarian failure. Premature ovarian failure is characterized by amenorrhea, infertility, and elevated gonadotropin levels in women under the age of 40. Because follistatin is essential for postnatal viability, we designed a cre/loxP conditional knockout system to render the follistatin gene null specifically in the granulosa cells of the postnatal ovary using Amhr2cre transgenic mice. The follistatin conditional knockout females develop fertility defects, including reduced litter number and litter sizes and, in the most severe case, infertility. Reduced numbers of ovarian follicles, ovulation and fertilization defects, elevated levels of serum FSH and LH, and reduced levels of testosterone were observed in these mice. These findings demonstrate that compromising granulosa cell follistatin function leads to findings similar to those characterized in premature ovarian failure. Follistatin conditional knockouts may therefore be a useful model with which to further study this human syndrome. These studies are the first report of a granulosa cell-specific deletion of a gene in the postnatal ovary and have important implications for future endeavors to generate ovary-specific knockout mouse models.

Central nervous system pathology in MFP2 deficiency: Insights from general and conditional knockout mouse models

Biochimie ◽  
2014 ◽  
Vol 98 ◽  
pp. 119-126 ◽  
Author(s):  
Simon Verheijden ◽  
Lien Beckers ◽  
Stephanie De Munter ◽  
Paul P. Van Veldhoven ◽  
Myriam Baes
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Mouse Models ◽  
Knockout Mouse ◽  
Conditional Knockout ◽  
Central Nervous System Pathology ◽  
Conditional Knockout Mouse

scholarly journals The Function of Selenium in Central Nervous System: Lessons from MsrB1 Knockout Mouse Models

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1372
Author(s):  
Tengrui Shi ◽  
Jianxi Song ◽  
Guanying You ◽  
Yujie Yang ◽  
Qiong Liu ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Synaptic Plasticity ◽  
Mouse Model ◽  
Mouse Models ◽  
Knockout Mouse ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Knockout Mouse Model ◽  
The Central Nervous System

MsrB1 used to be named selenoprotein R, for it was first identified as a selenocysteine containing protein by searching for the selenocysteine insert sequence (SECIS) in the human genome. Later, it was found that MsrB1 is homologous to PilB in Neisseria gonorrhoeae, which is a methionine sulfoxide reductase (Msr), specifically reducing L-methionine sulfoxide (L-Met-O) in proteins. In humans and mice, four members constitute the Msr family, which are MsrA, MsrB1, MsrB2, and MsrB3. MsrA can reduce free or protein-containing L-Met-O (S), whereas MsrBs can only function on the L-Met-O (R) epimer in proteins. Though there are isomerases existent that could transfer L-Met-O (S) to L-Met-O (R) and vice-versa, the loss of Msr individually results in different phenotypes in mice models. These observations indicate that the function of one Msr cannot be totally complemented by another. Among the mammalian Msrs, MsrB1 is the only selenocysteine-containing protein, and we recently found that loss of MsrB1 perturbs the synaptic plasticity in mice, along with the astrogliosis in their brains. In this review, we summarized the effects resulting from Msr deficiency and the bioactivity of selenium in the central nervous system, especially those that we learned from the MsrB1 knockout mouse model. We hope it will be helpful in better understanding how the trace element selenium participates in the reduction of L-Met-O and becomes involved in neurobiology.

scholarly journals Irradiation of Nf1 mutant mouse models of spinal plexiform neurofibromas drives pathologic progression and decreases survival

2021 ◽  
Author(s):  
Danny Laurent ◽  
Abbi E Smith ◽  
Waylan K Bessler ◽  
Marc Mendonca ◽  
Helen Chin-Sinex ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mouse Models ◽  
Conditional Knockout ◽  
Benign Neoplasms ◽  
Neoplastic Progression ◽  
Plexiform Neurofibromas ◽  
Normal Tissues ◽  
Peripheral Nerve Sheath Tumors ◽  
Conditional Knockout Mouse ◽  
Therapeutic Irradiation

Abstract Background Genetically susceptible individuals can develop malignancies after irradiation of normal tissues. In the context of therapeutic irradiation, it is not known whether irradiating benign neoplasms in susceptible individuals promotes neoplastic transformation and worse clinical outcomes. Individuals with Neurofibromatosis 1 (NF1) are susceptible to both radiation-induced second malignancies and spontaneous progression of plexiform neurofibromas (PNs) to malignant peripheral nerve sheath tumors (MPNSTs). The role of radiotherapy in the treatment of benign neoplasms such as PNs is unclear. Methods To test whether radiotherapy promotes neoplastic progression of PNs and reduces overall survival, we administered spinal irradiation (SI) to conditional knockout mouse models of NF1-associated PNs in two germline contexts: Nf1 fllfl; PostnCre + and Nf1 fl/-; PostnCre +. Both genotypes develop extensive Nf1 null spinal PNs, modeling PNs in NF1 patients. A total of 101 mice were randomized to 0 Gy, 15 Gy (3 Gy x 5), or 30 Gy (3 Gy x 10) of spine-focused, fractionated SI and aged until signs of illness. Results SI decreased survival in both Nf1 fllfl mice and Nf1 fl/- mice, with the worst overall survival occurring in Nf1 fl/- mice receiving 30 Gy. SI was also associated with increasing worrisome histologic features along the PN-MPNST continuum in PNs irradiated to higher radiation doses. Conclusions This pre-clinical study provides experimental evidence that irradiation of pre-existing PNs reduces survival and may shift PNs to higher grade neoplasms.

Sign in / Sign up

Export Citation Format

Share Document