Efficient removal ofloxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs

Abstract

The bacteriophage P1 Cre/loxP site‐specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP‐flanked DNA sequences in a gene‐targeted locus in fertilized eggs. We replaced a part of the T‐cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP‐flanked neogene in mouse embryonic stem (ES) cells by gene‐targeting technique. The resulting ES cell clones containing the mutant allele (VγLNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild‐type female mice. One hundred and seventy‐six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS‐Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre‐mediated recombination, resulting in a deletion of the loxP‐flanked sequences (VγΔ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the VγΔ locus to their offspring. The other 8 pups carried only wild‐type alleles. There were no pups carrying the unrecombined VγLNL locus. Thus, the frequency of Cre‐mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS‐Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the VγLNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997. © 1997 Wiley‐Liss, Inc.

γδ T cells impair airway macrophage differentiation in lung adenocarcinoma

“…The LSL-KRas G12D (Jackson et al, 2001), Rosa26 DM . lsl-MYC (Kruspig et al, 2018; RRID: IMSR_JAX:033805), Tcrd tm1Mom (Itohara et al, 1993), and Tcrg tm1Iku (Sunaga et al, 1997) alleles were previously described. All genetically modified mice were bred in house, maintained on a mixed FVB/N, C57BL/6 background, on a 12-hour light/dark cycle, and fed/watered ad libitum under specific pathogen-free conditions.…”

De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination

“…Amplification of ␥1 GLT was done by initial denaturing step of 94°C for 5 min, followed by 22 cycles of PCR (94°C for 30 s, 58°C for 30 s, 72°C for 1min) by using recombinant Taq polymerase (Takara) with a primer pair of I␥1F and C␥1R (11). Amplification of hypoxanthine-guanine phosphoribosyl transferase was initiated by a denaturing step of 94°C for 5 min, followed by 22 cycles of PCR (94°C for 30 s, 50°C for 30 s, 72°C for 1 min) by using recombinant Taq polymerase (Takara) with described primers (23).…”

A hallmark of active class switch recombination: Transcripts directed by I promoters on looped-out circular DNAs

“…Genomic DNA was purified from the same source after separation of RNA from TRIzol lysate. cDNA was synthesized with Superscript II (GIBCO) by using 2 g of total RNA and 1 g of poly d(T) [12][13][14][15][16][17][18] (Amersham Pharmacia) in a 20-l reaction volume, one twentieth of which was used as a template for reverse transcription (RT)-PCR in a 25-l reaction volume. Amplification of AID transcripts was done by an initial denaturing step of 94°C for 5 min followed by 22 cycles of PCR (94°C for 20 s, 58°C for 30 s, 72°C for 1 min) by using recombinant Taq polymerase (Takara) with a 119-and 118-primer pair (11).…”

“…CTs were detected after transfer to Hybond Nϩ membrane (Amersham Pharmacia) by a 32 P-labeled C P oligonucleotide probe located 41 bp upstream of the C R primer. Hypoxanthine-guanine phosphoribosyl transferase (HPRT) transcripts were amplified by 25 cycles of PCR (94°C for 30 s, 50°C for 30 s, 72°C for 1 min) by using recombinant Taq polymerase (Takara) with an HPRT-7 and HPRT-9 primer pair (17). ␣CD were PCR-amplified from 20 and 80 ng of genomic DNA from CH12F3-2A cells and splenocytes, respectively, by a denaturing step of 95°C for 1 min followed by 33 cycles at 98°C for 20 s and 68°C for 6 min by using LA Taq (Takara) with S␣F and S R primers in the presence of 2.5 mM Mg 2ϩ .…”