Generation of Cryab Knock-In Mice

Rosa26-UbiC- Cryab floxed mice were generated by Ozgene (Perth, WA, Australia). To generate the Cryab knock-in model, we designed a targeting vector containing a Flag-tagged Cryab cDNA (fl-Tg) preceded by a human ubiquitin C (UbiC) promoter as well as lox-P flanked polyadenylation (pA +) stop region, with a downstream flippase recombinase target site-flanked neomycin resistance cassette (PGK-NEO) for embryonic stem cell (ESC) selection. Genomic targeting of the construct was attained in ESCs of wild-type BALB/C, by utilizing standard homologous recombination and blastocyst manipulation techniques. Gene manipulation was validated by Ozgene using Southern blot analysis, with probes against both the endogenous coding region and NEO selection cassette following restriction digest of genomic DNA with the EcoRV restriction enzyme.

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Rashidieh B., Bain A.L., Tria S.M., Sharma S., Stewart C.A., Simmons J.L., Apaja P.M., Duijf P.H., Finnie J, & Khanna K.K. (2023). Alpha-B-Crystallin overexpression is sufficient to promote tumorigenesis and metastasis in mice. Experimental Hematology & Oncology, 12, 4.

Publication 2023

Blastocyst Cdna Embryonic stem cell Escs Gene Genomic Homologous recombination Human ubiquitin Mice Neomycin Polyadenylation Recombinase Restriction enzyme Southern blot analysis Ubiquitin c Vector

Corresponding Organization :

Other organizations : QIMR Berghofer Medical Research Institute, South Australian Health and Medical Research Institute, Queensland University of Technology, University of Adelaide

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Variable analysis

independent variables

Generation of the Cryab knock-in model using a targeting vector containing a Flag-tagged Cryab cDNA (fl-Tg) preceded by a human ubiquitin C (UbiC) promoter, a lox-P flanked polyadenylation (pA +) stop region, and a downstream flippase recombinase target site-flanked neomycin resistance cassette (PGK-NEO) for embryonic stem cell (ESC) selection

dependent variables

Validation of the gene manipulation by Southern blot analysis, with probes against both the endogenous coding region and NEO selection cassette following restriction digest of genomic DNA with the EcoRV restriction enzyme

control variables

Wild-type BALB/C embryonic stem cells used for genomic targeting of the construct

positive controls

None specified

negative controls

None specified

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