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

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.

Figure 1

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.

Figure 2

Pedigree analysis of the Kera-Cre transgenic mice mated with ZEG mice. Kera-Cre transgenic mice were crossbred with ZEG mice. Offspring from the mating male bitransgenic offspring (Kera-Cre/ZEG) with wild type C57BL6 mice, which carried the ZEG transgene, were whole body green regardless of the presence or absence of the Cre transgene. Symbols: squares, male; circles, female; dotted square and circle, bitransgenic mice with cornea-specific EGFP expression; filled square and circle, Z^∆EG mice with whole body EGFP expression. Non-transgenic animals were indicated with blank squares or circles. (A) Green fluorescence was observed in the cornea of a two-month-old Kera-Cre/ZEG mouse. (B) EGFP expression in E14.5 embryos from the timed mating of a male bitransgenic Kera-Cre/ZEG with a C57BL/6 female was photographed using white light (right) and UV light (left) under a ZEISS fluorescent stereomicroscope. In each photo, the animal on the left is the Z^∆EG embryo and the animal on the right is the wild type. Both photos showed identical animals (same embryo and same wild type animal) and just used different light sources to take each picture.

Figure 3

Pedigree analysis of the Kera-Cre transgenic mice mated with Rosa26R homozygous (R26R) mice. Squares stood for males, and circles stood for female animals; mosaic circles and squares indicate female and male animals, respectively, with a tissue specific manner of expressing Cre in the corneal stroma. The solid squares and circles indicate male and female animals, respectively, with expressing whole body blue by X-gal staining. Genotypes of experimental mice were determined by PCR of tail DNA as indicated under each of individual mice. Cre or R26R ^f/w indicates single heterozygous transgenic mice harboring the Cre or Rosa26R gene alone; Cre/Rsa26R^f/w indicates double transgenic mice harboring both Cre and heterozygous Rosa26R alleles and a wild type (w) Rosa allele. Non-transgenic animals were indicated with open squares or circles. (A) Detection of Cre-mediated recombination in the corneal stroma from double transgenic Kera-Cre/Rosa26R^f/w mice is shown. Whole-mount X-gal staining was performed on the eye and hind limb from a two-month-old double transgenic Kera-Cre/Rosa26R^f/w mouse. (B) X-gal staining in E14.5 wild type (WT) embryo and transgenic embryos with excision of the reporter gene is shown. Embryos were collected at E14.5 from C57BL/6 female mated with male double transgenic Kera-Cre/R26R mouse. The β-gal activity was detected by whole-mount X-gal staining.

Figure 4

Reverse transcription polymerase chain reaction for detection of keratocan, keratin 12 and Cre mRNAs in testis. Total RNAs were isolated from four pooled corneas and individual testis of two Kera-Cre mice with TRIzol Reagent (Invitrogen), and then the total RNAs were dissolved in DEPC-Water and stored at −80 °C until use. Ten micrograms (testis) and 3.5 μg of total RNA were annealed to oligo-dT and reverse transcribed with kits from Promega according to the manufacturer’s instruction. Single stranded cDNA was subjected to PCR reactions using primer pairs for keratocan (Kera), keratin 12 (K12), Cre recombinase (Cre), and glyceraldehydes phosphate dehydrogenase (GAPDH) as described in Methods. A: Use of primers in exons 1 and 3 of Krt12 generates a 408 bp RT–PCR product with total RNA from the cornea, but not that from testis; B: use of primers in exons 7 and 8 of Krt12, a 410 bp RT–PCR product was detected with RNA from both corneas and testis; C: use of primers in exons 2 and 3 of Kera produces a 350 bp RT–PCR product with RNA from cornea and testis; D, Use primer of exon 1 of Kera and primer of IRES yields a 480 bp RT–PCR product in RNA from both cornea and testis of Kera-Cre mice; E: primers of exon 6 and 7 of GAPDH gene, a 200 bp transcript was used as a positive control RT–PCR with RNA from both cornea and testis. Lane 1, testis 1; lane 2, testis 2; lane 3, cornea; lane 4, control (no cDNA added); M, 1 kb DNA markers.

Figure 5

Expression of the LacZ gene in double transgenic Kera-Cre/Rosa26R mice during spermatogenesis. Whole mount X-gal staining of testes collected from 20-(A), 30-(B), and 60 day-old (C) bitransgenic mice showed positive reaction in primary and secondary spermatocytes and spermatids. No β-gal activity was found in adult single Rosa26R transgenic testes and 10-day-old testes of double Kera-Cre/Rosa26R mice (data not shown). Leydig cells (arrowheads) have endogenous β-gal activity. Testicular cell suspensions prepared from a Kera-Cre/Rosa26R mouse were subjected to May–Grünwald-Giemsa stain after X-gal staining. Positive X-gal staining (arrow) was observed in the primary spermatocytes (D), primary spermatocytes in meiotic division (E), spermatids (F), and round spermatids (G).

Figure 6

Cre-mediated recombination of the Smad4^f allele in spermatogenic cells of double transgenic Kera-Cre/Smad4^f/wt mouse. (A) Histograms of flow cytometry of testicular cell suspension obtained from a 60-day-old double transgenic Kera-Cre/Smad4^f/w mouse showed a distribution of 65%, 9%, and 17% for 1N, 2N, and 4N cells, respectively. PCR of genomic DNA from 1N and 4N cells showed excision of the Smad4^f allele but not from 2N cells. (B) Histogram showed enrichment of 2N cells by Percoll gradients as described in Methods. PCR analysis of these enriched 2N cells demonstrated excision of the Smad4^f allele. Primers b and c identify wild type Smad4 (390 bp) and floxed Smad4^f allele (450 bp) whereas primers a and c identify the excised Smad4^f allele (500 bp). TC, testicular cells; M, DNA marker.

Figure 7

Hypothetical cryptic Cre activity during gametogenesis. It is hypothesized that the expression of Cre occurs when progenitor germ cells commit terminal differentiation and enter gametogenesis at which time floxed allele(s) may undergo Cre-mediated recombination.