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Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice.

Weng DY, Zhang Y, Hayashi Y, Kuan CY, Liu CY, Babcock G, Weng WL, Schwemberger S, Kao WW - Mol. Vis. (2008)

Bottom Line: Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis.Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion.Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Cincinnati, Cincinnati, OH 45267-0838, USA.

ABSTRACT

Purpose: To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice.

Methods: Cre driver mice were crossbred with reporter mouse lines (e.g., ZEG and Rosa26R) to obtain Cre/ZEG and Cre/Rosa26R double transgenic mice. The frequency of promiscuous LoxP/Cre recombination was determined by the expression of second reporter genes in the offspring of double transgenic mice.

Results: The frequency of promiscuous LoxP/Cre recombination varied in different lines of Cre driver mice and in the sex of the same driver mice with higher penetrance in male than in female double transgenic mice. Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis. Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion.

Conclusions: Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

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Related in: MedlinePlus

Diagram of KC (Kera-Cre) minigene construct. An expression DNA construct was prepared with pBskII plasmid (Strategen, La Jolla, CA) using conventional cloning strategy, which contained 3.2 kb of 5′ DNA fragment upstream of the transcription initiation site, 172 bp of exon 1, the full length of intron 1 (1.4 kb), and 7 bp of exon 2 preceding the ATG starting codon of Kera, and an IRES-NLS-Cre minigene. The minigene was released by Not1 and Fsp1 restriction enzyme digestion and used in the microinjection of fertilized eggs by Transgenic Core at the Children’s Hospital of Cincinnati.
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f1: Diagram of KC (Kera-Cre) minigene construct. An expression DNA construct was prepared with pBskII plasmid (Strategen, La Jolla, CA) using conventional cloning strategy, which contained 3.2 kb of 5′ DNA fragment upstream of the transcription initiation site, 172 bp of exon 1, the full length of intron 1 (1.4 kb), and 7 bp of exon 2 preceding the ATG starting codon of Kera, and an IRES-NLS-Cre minigene. The minigene was released by Not1 and Fsp1 restriction enzyme digestion and used in the microinjection of fertilized eggs by Transgenic Core at the Children’s Hospital of Cincinnati.

Mentions: Figure 1 showed the minigene construct used to create Kera-Cre (KC) driver mice. The minigene contains a 3.2 kb DNA fragment 5′ of the transcription initiation site of Kera, exon 1 (172 bp), intron 1 (1.4 kb), exon 2 (7 bp) preceding the ATG starting codon of Kera mRNA, and a Cre minigene with IRES (internal ribosome entry site) and NLS (nuclear localization sequences) followed by a SV40 polyadenylation signal [4]. The minigene was released from the plasmid by Not1 and Fsp1 restriction enzyme digestion and used in microinjection of fertilized FVB/N eggs. Twenty-three independent transgenic mouse lines (F0) were obtained and crossed with Rosa26R reporter mice [3]. Functional F1 offspring from each transgenic line was identified by X-gal staining. Six founders did not transmit the transgene to their offspring by polymerase chain reaction (PCR) genotype; six lines did not express the Cre minigene and failed to recombine the R26R allele to express the LacZ reporter gene. These mouse lines were not examined further. Eleven transgenic founder lines harboring the Kera-Cre transgene were divided into three categories based on the population of βgal (β-galactosidase) positive cells in the cornea. Three mouse lines showed very low numbers of X-gal positive cells in the cornea. Five founder lines showed moderate positive cells, and three mouse lines showed very high numbers of positive cells in the cornea. To further verify the ocular tissues that express the transgene, the histological analysis of whole-mount X-gal stained eyes confirms that X-gal positive cells are found in the corneal stroma and not in the corneal epithelium and endothelium. One strong line (KC4.3) and one moderate line (KC4.1) were used in present studies. Both lines show the same promiscuous recombination of LoxP modified alleles during gametogenesis. Detailed cell lineage analyses using these Cre drivers are in progress and will be reported in the future (manuscript in preparation).


Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice.

Weng DY, Zhang Y, Hayashi Y, Kuan CY, Liu CY, Babcock G, Weng WL, Schwemberger S, Kao WW - Mol. Vis. (2008)

Diagram of KC (Kera-Cre) minigene construct. An expression DNA construct was prepared with pBskII plasmid (Strategen, La Jolla, CA) using conventional cloning strategy, which contained 3.2 kb of 5′ DNA fragment upstream of the transcription initiation site, 172 bp of exon 1, the full length of intron 1 (1.4 kb), and 7 bp of exon 2 preceding the ATG starting codon of Kera, and an IRES-NLS-Cre minigene. The minigene was released by Not1 and Fsp1 restriction enzyme digestion and used in the microinjection of fertilized eggs by Transgenic Core at the Children’s Hospital of Cincinnati.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2274927&req=5

f1: Diagram of KC (Kera-Cre) minigene construct. An expression DNA construct was prepared with pBskII plasmid (Strategen, La Jolla, CA) using conventional cloning strategy, which contained 3.2 kb of 5′ DNA fragment upstream of the transcription initiation site, 172 bp of exon 1, the full length of intron 1 (1.4 kb), and 7 bp of exon 2 preceding the ATG starting codon of Kera, and an IRES-NLS-Cre minigene. The minigene was released by Not1 and Fsp1 restriction enzyme digestion and used in the microinjection of fertilized eggs by Transgenic Core at the Children’s Hospital of Cincinnati.
Mentions: Figure 1 showed the minigene construct used to create Kera-Cre (KC) driver mice. The minigene contains a 3.2 kb DNA fragment 5′ of the transcription initiation site of Kera, exon 1 (172 bp), intron 1 (1.4 kb), exon 2 (7 bp) preceding the ATG starting codon of Kera mRNA, and a Cre minigene with IRES (internal ribosome entry site) and NLS (nuclear localization sequences) followed by a SV40 polyadenylation signal [4]. The minigene was released from the plasmid by Not1 and Fsp1 restriction enzyme digestion and used in microinjection of fertilized FVB/N eggs. Twenty-three independent transgenic mouse lines (F0) were obtained and crossed with Rosa26R reporter mice [3]. Functional F1 offspring from each transgenic line was identified by X-gal staining. Six founders did not transmit the transgene to their offspring by polymerase chain reaction (PCR) genotype; six lines did not express the Cre minigene and failed to recombine the R26R allele to express the LacZ reporter gene. These mouse lines were not examined further. Eleven transgenic founder lines harboring the Kera-Cre transgene were divided into three categories based on the population of βgal (β-galactosidase) positive cells in the cornea. Three mouse lines showed very low numbers of X-gal positive cells in the cornea. Five founder lines showed moderate positive cells, and three mouse lines showed very high numbers of positive cells in the cornea. To further verify the ocular tissues that express the transgene, the histological analysis of whole-mount X-gal stained eyes confirms that X-gal positive cells are found in the corneal stroma and not in the corneal epithelium and endothelium. One strong line (KC4.3) and one moderate line (KC4.1) were used in present studies. Both lines show the same promiscuous recombination of LoxP modified alleles during gametogenesis. Detailed cell lineage analyses using these Cre drivers are in progress and will be reported in the future (manuscript in preparation).

Bottom Line: Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis.Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion.Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Cincinnati, Cincinnati, OH 45267-0838, USA.

ABSTRACT

Purpose: To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice.

Methods: Cre driver mice were crossbred with reporter mouse lines (e.g., ZEG and Rosa26R) to obtain Cre/ZEG and Cre/Rosa26R double transgenic mice. The frequency of promiscuous LoxP/Cre recombination was determined by the expression of second reporter genes in the offspring of double transgenic mice.

Results: The frequency of promiscuous LoxP/Cre recombination varied in different lines of Cre driver mice and in the sex of the same driver mice with higher penetrance in male than in female double transgenic mice. Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis. Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion.

Conclusions: Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

Show MeSH
Related in: MedlinePlus