Dataset Information

Highly reliable creation of floxed alleles by electroporating single-cell embryos.

ABSTRACT:

Background

Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking.

Results

Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here).

Conclusions

Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

SUBMITTER: Sentmanat MF

PROVIDER: S-EPMC8815186 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Highly reliable creation of floxed alleles by electroporating single-cell embryos.

Sentmanat Monica F MF White J Michael JM Kouranova Evguenia E Cui Xiaoxia X

BMC biology 20220204 1

<h4>Background</h4>Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking.<h4>Results</h4>Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specifi ...[more]

PMID: 35115009

Similar Datasets

Project description:Establishment and subsequent validation of a aCGH protocol for WGA (whole genome amplification) products originating from single cell or low amount of starting material (i.e. microdissected FFPE tissue samples). The establishment of the protocol involved testing of three DNA labeling protocols. Two labeling protocols were designed specifically for Ampli1(TM) WGA products. Additionally random primed isothermal (Klenow-based) labeling approach was tested (MM-CM-6hlendick et al., PLoS One. 2013 Jun 25;8(6):e67031.). In addition two different types of reference samples were tested and reference based on single-cell WGA products was chosen as most suitable in the end. The validation of the protocol assessed the following aspects: (1) performance of the protocol on primary and reamplified WGA products, (2) accuracy of the protocol in term of sensitivity of the CNA detection, (3) accuracy in terms of recapitulation of complex patterns of CNAs, (4) accuracy in terms of quantitative assessment of the CNAs, (5) ability to detect genomic heterogeneity of single cells (obtained either from in vitro cultures or from clinical patient material), (6) ability to detect minimal regions of aberration within a panel of disseminated cancer cells and corresponding tumor tissues. Establishment and validation of the single-cell aCGH protocol: two condition experiment (i.e. PCR-based labeling technique 1 vs. PCR-based labeling technique 2; PCR-based labeling technique 2 vs. random-primed isothermal (Klenow) labeling approach; reference DNA from cell pool WGA product vs. reference DNA from single-cell WGA products). Validation of the protocol: comparison of the CNA profiles between single-cell WGA products and corresponding bulk DNA. Analysis of the DCC and corresponding FFPE tumor tissue samples: single-condition experiment performed on samples collected at different stages of the disease (DCCs from bone marrow) and/or from different sites (primary tumor-breast; metastasis-lymph nodes; DCCs-bone marrow). Microarray data is corresponding data depicted in the paper manuscript titled: Reliable single cell array CGH for clinical samples

Dataset Information

Highly reliable creation of floxed alleles by electroporating single-cell embryos.

Background

Results

Conclusions

Publications

Highly reliable creation of floxed alleles by electroporating single-cell embryos.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets