Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells

Abstract

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-f lanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.

Systemic inactivation of Hs6st1 in mice is associated with late postnatal mortality without major defects in organogenesis

“…2). In PrmCre transgenic males, the protamine 1 (Prm1) promoter drives Cre recombinase expression specifically in the germline, whereas in Zp3Cre transgenic females, the zona pellucida 3 (Zp3) promoter drives Cre expression specifically in maturing oocytes (deVries et al, 2000;O'Gorman et al, 1997). Thus, these initial crosses generated Zp3Cre Tg/1; Hs6st1 flox/1 females and PrmCre Tg/1; Hs6st1 flox/1 males.…”

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

“…To examine how EGFP‐Centrin1 was regulated during cell cycle, a mouse line able to express EGFP‐Centrin1 in all cell types under control of the ROSA locus was generated utilizing Protamine Cre to ablate the Neo‐polyA cassette in ROSA‐ Neo ‐EGFP‐Cetn1 mice (O'Gorman et al ., 1997). ROSA‐EGFP‐Cetn1 mice were maintained for more than 22 generations, bred and grew well, with no overt phenotypes.…”

“…Protamine‐Cre (O'Gorman et al ., 1997), Nkx2.5‐Cre Troponin T‐Cre (Jiao et al ., 2003), and R26‐mTmG (Muzumdar et al ., 2007) were purchased from Jackson Laboratories. ROSA‐ Neo ‐EGFP‐Cetn1 line have been bred into a Black Swiss outbred background for more than 6 generations, did not show any overt difference from wild‐type mouse.…”

Human CD22 cannot fully substitute murine CD22 functions in vivo, as shown in a new knockin mouse model

“…By in-frame knockin of the cDNA of human CD22 after the coding sequence for the signal peptide of murine Cd22, we wanted to ensure that the human CD22 will be transported normally to the B-cell surface. E14CreAg Embryonic Stem (ES) cells [15], were transfected with the targeting vector and ES cells were screened by PCR and positive clones confirmed by Southern blot (Supporting Information Fig. 1).…”

“…The targeting vector was opened at the short arm by digesting it with the enzyme NotI and the DNA was then sterilized by ethanol precipitation. The embryonic stem cells from the line E14CreAG (129sv background, expressing protamine-Cre) [15] were transfected with the linearized vector by electroporation. Clones were screened for homologous integration by PCR and verified by Southern blot with an external probe generated with the primers humCD22Sonde3 (5 -CCT CTG TGG GAT TGA CCT TG-3 ) and humCD22Sonde4 (5 -CCT CTG ACC ATA GTC ACC TG-3 ) and with an internal probe generated by digesting the external probe with the enzyme EcoRI and isolating the 720 bp band.…”