The subpopulation of CF‐1 mice deficient in P‐glycoprotein contains a murine retroviral insertion in the <i>mdr1a</i> gene

Pippert, Todd R.; Umbenhauer, Diane R.

doi:10.1002/jbt.3

Cited by 31 publications

(26 citation statements)

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“…The Mdr1a ZFNs target exon 7, which encodes TMs 3 and 4. On the basis of previous work in this field, any partial protein that might result from the described frameshift and nonsense mutations we observed (assuming such protein fragments could be stable) should not be functional (Pippert and Umbenhauer 2001). Among the mutant alleles, 41% cause exon skipping, 37% result in frameshift, and the rest carry in-frame deletions (Table S1).…”

Section: Discussionmentioning

confidence: 84%

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Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

et al. 2010

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Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to .1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research. C ONVENTIONAL gene targeting technology in mice relies on homologous recombination in embryonic stem (ES) cells to target specific gene sequences, most commonly to disrupt gene function (Doetschman et al. 1987;Kuehn et al. 1987;Thomas and Capecchi 1987). Advantages of gene targeting in ES cells are selective target sequence modification, the ability to insert or delete genetic information, and the stability of the targeted mutations through subsequent generations. There are also potential limitations, including limited rates of germline transmission and strain limitations due to lack of conventional ES cell lines (Ledermann 2000;Mishina and Sakimura 2007). Moving the targeted allele from one strain to another requires 10 generations of backcrosses that take 2-3 years. A minimum of 1 year is necessary for backcrossing if speed congenics is applied (Markel et al. 1997).Zinc-finger nucleases (ZFNs) are fusions of specific DNA-binding zinc finger proteins (ZFPs) and a nuclease domain, such as the DNA cleavage domain of a type II endonuclease, FokI (Kim et al. 1996;Smith et al. 1999;Bibikova et al. 2001). A pair of ZFPs provide target specificity, and their nuclease domains dimerize to cleave the DNA, generating double strand breaks (DSBs) (Mani et al. 2005), which are detrimental to the cell if left unrepaired (Rich et al. 2000). The cell uses two main pathways to repair DSBs: high-fidelity homologous recombination and error-prone nonhomologous end joining (NHEJ) (Lieber 1999;Pardo et al. 2009;Huertas 2010). ZFN-mediated gene disruption results from deletions or insertions frequently introduced by NHEJ. Figure 1 illustrates the cellular events following the injec...

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Section: Discussionmentioning

confidence: 84%

“…All 12 TM domains as well as the two ATP-binding motifs are essential for Mdr1a function (Pippert and Umbenhauer 2001). The Mdr1a ZFNs target exon 7, which encodes TMs 3 and 4.…”

Section: Discussionmentioning

confidence: 99%

Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

et al. 2010

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“…Animal studies were conducted under protocols approved by the St. Jude Children's Research Hospital Committee on the Use and Care of Animals. Female (12-to 16-week-old) P-glycoprotein (P-gp) wild-type (WT) and knockout (KO) [CF-1 mice (Charles River Laboratories, Wilmington, MA) deficient in Pgp] (Pippert and Umbenhauer, 2001) and C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were maintained in a pathogen-free facility. Abcg2-EGFP (enhanced green fluorescent protein) mice have been previously described (Tadjali et al, 2006).…”

Section: Methodsmentioning

confidence: 99%

Drug Transporters on Arachnoid Barrier Cells Contribute to the Blood–Cerebrospinal Fluid Barrier

et al. 2013

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The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tightjunctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.

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“…Therefore, the role of P-gp in the brain distribution of abacavir was evaluated in vivo using the CF1 mouse model. The mdr1a(Ϫ/Ϫ) CF1 mouse model is a subpopulation naturally deficient in the expression of the murine mdr1a gene product, due to the loss of exon 23 during mRNA splicing (Pippert and Umbenhauer, 2001). These mutant mice lack functional P-gp in tissues such as the brain, intestine, and testis (Umbenhauer et al, 1997) and are thus useful in understanding the role of P-gp in limiting CNS distribution (Kwei et al, 1999).…”

Section: Fig 8 a Directional Flux Of [mentioning

confidence: 99%

P-glycoprotein-Mediated Active Efflux of the Anti-HIV1 Nucleoside Abacavir Limits Cellular Accumulation and Brain Distribution

Shaik¹,

Giri

Pan

et al. 2007

Drug Metab Dispos

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ABSTRACT:P-glycoprotein (P-gp)-mediated efflux at the blood-brain barrier has been implicated in limiting the brain distribution of many anti-HIV1 drugs, primarily protease inhibitors, resulting in suboptimal concentrations in this important sanctuary site. The objective of this study was to characterize the interaction of abacavir with P-gp and determine whether P-gp is an important mechanism in limiting abacavir delivery to the central nervous system (CNS). In vitro and in vivo techniques were employed to characterize this interaction. Abacavir stimulated P-gp ATPase activity at high concentrations. The cellular accumulation of abacavir was significantly decreased by ϳ70% in Madin-Darby canine kidney II (MDCKII)-MDR1 monolayers compared with wild-type cells and was completely restored by the P-gp inhibitors ((R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo(a,e)cyclopropa(c)cycloheptan-6-yl)-␣-((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride) (LY335979) and N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918). Directional flux experiments indicated that abacavir had greater permeability in the basolateral-to-apical direction (1.58E-05 cm/s) than in the apical-to-basolateral direction (3.44E-06 cm/s) in MDR1-transfected monolayers. The directionality in net flux was abolished by both LY335979 and GF120918. In vivo brain distribution studies showed that the AUC plasma in mdr1a(؊/؊) CF-1 mutant mice was ϳ2-fold greater than the AUC plasma in the wild type, whereas the AUC brain in the mutant was 20-fold higher than that in the wild type. Therefore, the CNS drug targeting index, defined as the ratio of AUC brain-to-plasma for mutant over wild type, was greater than 10. These data are the first in vitro and in vivo evidence that a nucleoside reverse transcriptase inhibitor is a P-gp substrate. The remarkable increase in abacavir brain distribution in P-gp-deficient mutant mice over wild-type mice suggests that P-gp may play a significant role in restricting the abacavir distribution to the CNS.Abacavir, a carbocyclic nucleoside and a guanosine analog, is a member of the nucleoside reverse transcriptase (NRTI) family of anti-HIV1 agents. Abacavir is metabolized intracellularly to its active form, carbovir triphosphate (Daluge et al., 1997). Abacavir is used in combination with other NRTIs as well as protease inhibitors in active retroviral therapy (ART) (Josephson et al., 2007).HIV1 can infect the CNS at a very early stage of the disease and can lead to the development of AIDS-dementia complex (Ances and Ellis, 2007). The CNS has been cited as a reservoir for the HIV1 virus due to incomplete suppression of the viral replication (Kerza-Kwiatecki and Amini, 1999). Suboptimal concentrations of anti-HIV1 drugs in the CNS can not only lead to incomplete eradication of the virus but also provide ideal conditions for the selection of more virulent mutants (Lipniacki, 2003). Recent reports have highlighted the compartmentalizati...

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The subpopulation of CF‐1 mice deficient in P‐glycoprotein contains a murine retroviral insertion in the mdr1a gene

Cited by 31 publications

References 18 publications

Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

Drug Transporters on Arachnoid Barrier Cells Contribute to the Blood–Cerebrospinal Fluid Barrier

P-glycoprotein-Mediated Active Efflux of the Anti-HIV1 Nucleoside Abacavir Limits Cellular Accumulation and Brain Distribution

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