Overview

These OVE2353A-SB1 mice harbor a mutation created by random insertion of the SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229), and the transposon was subsequently mobilized via exposure to sleeping beauty transposase (SB10) to generate the mutation. The donating investigator reports the phenotype of homozygous mice as: flattened "pancake" appearance at embryonic day (E)9.

Donating Investigator

Paul A Overbeek, Baylor College of Medicine

Genetic overview

Genetic Background	Generation

In(18E2;Smad4)3Ove

Allele Type	Gene Symbol	Gene Name
Transposon induced	In(18E2;Smad4)3Ove	inversion, Chr 18, Paul A Overbeek 3

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Research Applications

Developmental Biology Research

View All Research Applications

Details

Detailed Description

These OVE2353A-SB1 mice harbor a mutation created by random insertion of the SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229), and the transposon was subsequently mobilized via exposure to sleeping beauty transposase (SB10) to generate the mutation. Using inverse PCR analysis, a chromosomal inversion was identified on chromosome 18 between the original lentiviral integration site (agenic region linked to the (-) strand of DNA at position 86,038,197 bp [NCB137/mm9; 3'-86,038,197(-)]) and the transposition site (intron 9 of the Smad4 gene (Smad4); the left IR/DR is linked to the (-) strand of DNA at position 73,805,670 bp [NCB137/mm9; L3-73,805,670(-)]). The donating investigator reports the phenotype of homozygous mice as: flattened "pancake" appearance at embryonic day (E)9.

Development

A lentiviral transgenic approach was used to generate these mice. The SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229) was designed with a Sleeping Beauty (SB) transposon (inward-facing SB arms flanking a tandem pair of cHS4 core insulator elements), followed by a mouse tyrosinase minigene (Tyro) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in the FUGW self-inactivating HIV-based lentiviral vector backbone. The SB transposon and Tyro minigene replaced the ubiquitin-c promoter and EGFP sequences originally found in the FUGW lentiviral vector. The SB transposon used in this transgene has an inverted repeat/direct repeat sequence (IR/DR; the SB transposon recognition site), two copies of the 0.25 kb chicken beta-globin core insulator element (cHS4 core), and a second IR/DR sequence. The IR/DR sequences are inward-facing (pointed towards the cHS4core). The cHS4 core insulator element functions to terminate transcription of the upstream locus into which it integrates (and also enhance expression from the downstream Tyro minigene). The Tyro minigene is composed of the mouse Tyr enhancer region (623 bp), promoter region (657 bp), and 1566 bp cDNA sequence (including the stop codon); all in sense orientation relative to the FUGW backbone. There is no polyA site between the Tyro minigene and WPRE sequence. The WPRE sequence functions to enhance the mRNA transcript stability. This packaged lentiposon was injected subzonally (under the zona pellucida) into 1-8 cell stage FVB/N mouse embryos. Expression of the tyrosinase minigene results in melanin synthesis, so founder mice (F0) were identified by inspection for pigmentation. All F0 mice exhibited mosaic pigmentation. Most F0 mice had more than one lentiviral integration site. F0 mice were assigned an OVE number and then bred with FVB/N mice. Pigmented offspring (F1) with different coat colors were designated as subline A, B, C, etc., for each family. F1 mice were bred with FVB/N mice. F2 mice with different coat colors were designated as sublines A-1, A-2, etc., or B-1, B-2, etc. Breeding with FVB/N mice was continued until litters with only one or two different coat colors were obtained. Mice with identical coat colors were then inbred to generate homozygotes. Homozygous mice were viable and fertile. Homozygous females were bred to males with testes-specific expression of the SB transposase (PGK2-SB10 transgenic line OVE1780 on an FVB/N genetic background). The resulting double mutant male offspring ("seed males") have the ability to mobilize transposons in their germline; and mobilization from the inward-facing, IR/DR configuration leads to genomic rearrangements including deletions, inversions and translocations, in conjunction with loss of the cHS4 core insulator element initially located between the IR/DR elements. The seed males were bred with FVB/N wildtype females, and the offspring were screened for deletion of the core insulator element. Mice with transpositions (new F0 mice) were bred to FVB/N mice to establish new lines (SB1, SB2, etc.). F1 mice for each new line were inbred and assessed for the presence or absence of viable homozygotes. Hemizygous mice of line OVE2353A-SB1 (OVE#2353A-SB1) exhibit medium grey coat color. The donating investigator reports the PGK2-SB10 transgene was bred out of the line as well. Using inverse PCR analysis, a chromosomal inversion was identified on chromosome 18 between the original lentiviral integration site (agenic region linked to the (-) strand of DNA at position 86,038,197 bp [NCB137/mm9; 3'-86,038,197(-)]) and the transposition site (intron 9 of the Smad4 gene (Smad4); the left IR/DR is linked to the (-) strand of DNA at position 73,805,670 bp [NCB137/mm9; L3-73,805,670(-)]). Transgenic males were used to freeze sperm, and that cryopreserved sperm were sent to MMRRC-JAX. Upon arrival, aliquots of frozen sperm used to fertilize FVB/NJ embryos.

Control Suggestions

Wild-type from the colony
001800 FVB/NJ

Additional Information

Considerations for Choosing Controls

Genetics

In(18E2;Smad4)3Ove

Allele Symbol: In(18E2;Smad4)3Ove

Allele Name	inversion, Chr 18, Paul A Overbeek 3
Allele Type	Transposon induced
Allele Synonym(s)	In(18)2353A-SB1Ove; In3Ove; OVE#2353A-SB1
Gene Symbol and Name	In(18E2;Smad4)3Ove, inversion, Chr 18, Paul A Overbeek 3
Gene Synonym(s)
Strain of Origin	FVB/N
Chromosome	18
Molecular Note	The SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229) was designed with a Sleeping Beauty (SB) transposon (inward-facing SB arms flanking a tandem pair of cHS4 core insulator elements), followed by a mouse tyrosinase minigene (Tyro) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in the FUGW self-inactivating HIV-based lentiviral vector backbone. The SB transposon and Tyro minigene replaced the ubiquitin-c promoter and EGFP sequences originally found in the FUGW lentiviral vector. The SB transposon used in this transgene has an inverted repeat/direct repeat sequence (IR/DR; the SB transposon recognition site), two copies of the 0.25 kb chicken beta-globin core insulator element (cHS4 core), and a second IR/DR sequence. The IR/DR sequences are inward-facing (pointed towards the cHS4core). The cHS4 core insulator element functions to terminate transcription of the upstream locus into which it integrates (and also enhance expression from the downstream Tyro minigene). The Tyro minigene is composed of the mouse Tyr enhancer region (623 bp), promoter region (657 bp), and 1566 bp cDNA sequence (including the stop codon); all in sense orientation relative to the FUGW backbone. There is no polyA site between the Tyro minigene and WPRE sequence. The WPRE sequence functions to enhance the mRNA transcript stability. The transposon was mobilized by SB10. In line OVE2353A-SB1, lentiviral integration leads to an inversion between the original integration site an agenic region in chromosome 18 E2 [NCBI37/mm9; 3'-86,038,197(-)] and intron 9 of Smad4 [NCBI37/mm9; L3-73,805,670(-)].

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Developmental Biology Research

Mammalian Phenotype Terms by Genotype

The following phenotype relates to a compound genotype created using this strain

Genotype: In(18E2;Smad4)3Ove/In(18E2;Smad4)3Ove
involves: FVB/N

embryo phenotype

abnormal embryo development
- flattened "pancake" appearance at E9

References

Additional - In(18E2;Smad4)3Ove related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Separated PCR:In(18E2;Smad4)3Ove-3'-1
- Separated MCA:In(18E2;Smad4)3Ove 3'-1
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, heterozygous mice may be bred together, bred with wildtype siblings, or bred with FVB/N inbred mice.
- Additional Breeding and Husbandry Support
Citation
When using the FVB/N-In(18E2;Smad4)3Ove/Mmjax mouse strain in a publication, please cite the originating article(s) and include MMRRC stock #36255 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Terms Of Use

General Terms and Conditions

See MMRRC for Additional Conditions of Distribution

Questions about Terms of Use

Licensing Information

Phone: 207-288-6470

Email: TechTran@jax.org

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Donating Investigator

Genetic overview

Research Applications

Detailed Description

Development

Control Suggestions

Additional Information

In(18E2;Smad4)3Ove

Allele Symbol: In(18E2;Smad4)3Ove

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

Genotype: In(18E2;Smad4)3Ove/In(18E2;Smad4)3Oveinvolves: FVB/N

embryo phenotype

References

Additional - In(18E2;Smad4)3Ove related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

The Jackson Laboratory's Genotype Promise

Terms Of Use

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Genotype: In(18E2;Smad4)3Ove/In(18E2;Smad4)3Ove
involves: FVB/N