Mouse unable to express functional alpha-4 integrin protein, and methods for assaying compounds or agents for alpha-4 integrin protein antagonist activity and a genetic marker for evaluating efficacy of modulators of signaling activity of a VLA-4 receptor
OwnerMonika Wasel-Nielen, Bernhard Kirschbaum +4inventors
InventorsMonika Wasel-Nielen, Bernhard Kirschbaum, Martyn Foster, Gregory Polites, Olga Khorkova, Bin Zhu
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Abstract
Provided herein is a mouse that is unable to express functional alpha-4 integrin protein, and methods for assaying agents for alpha-4 integrin antagonist activity, as well genetic markers for analyzing the efficacy of VLA-4 modulators, and particularly antagonists.
Claims54
A mouse that is unable express functional alpha-4 integrin protein.
A knockout mouse that is unable to express functional alpha-4 integrin protein, wherein said knockout mouse has a genome comprising:
- (a) first and second alleles capable of expressing functional alpha-4 integrin protein that have defects that prevent the alleles from expressing functional alpha-4 integrin protein;
- and
- (b) two copies of a transgene comprising a DNA sequence of SEQ ID NO:
- 1, wherein said knockout mouse is unable to express functional alpha-4 integrin protein.
A method for making a knockout mouse that is unable to express functional alpha-4 integrin protein, comprising the steps of:
- (a) crossing two knockout mice comprising a first and second allele capable of expressing functional alpha-4 integrin protein, wherein the knockout mice each comprise a defect in either the first allele or second allele, such that either the first or second allele in each knockout mouse is unable to express functional alpha-4 integrin protein;
- (b) harvesting embryos resulting from the cross of step (a), wherein the embryos are heterozygous for the defect;
- (c) inserting a transgene comprising a portion of an isolated cDNA molecule that encodes for alpha-4 integrin protein operatively associated with a promoter, into each embryo harvested in step (b), to form a transfected embryo;
- (d) inserting the transfected embryo into a pseudopregnant female mouse so that the pseudopregnant female mouse gives birth to a mouse whose genome comprises:
- (i) first and second alleles capable of expressing functional alpha-4 integrin protein, wherein either the first or the second allele comprises the defect that prevents the allele from expressing functional alpha-4 integrin protein, and
- (ii) the transgene integrated into said genome;
- and
- (e) crossing two mice produced in step (d) to produce an alpha-4 homozygous knockout mouse whose genome comprises two copies of the transgene,
- wherein the resulting knockout mouse of step (e) is unable to express functional alpha-4 integrin protein.
A method for making a knockout mouse that is unable to express functional alpha-4 integrin protein, comprising the steps of:
- (a) crossing two heterozygous alpha-4 integrin knockout mice assigned Jackson laboratories stock number 002463;
- (b) harvesting the embryos that result from the cross of step (a);
- (c) inserting a transgene-comprising a DNA sequence of SEQ ID NO:
- 1 into each embryo harvested in step (b), to form a transfected embryo;
- (d) inserting the transfected embryo into a pseudopregnant female mouse so that the pseudopregnant female mouse gives birth to an alpha-4 integrin heterozygous knockout mouse having the transgene within its genome;
- and
- (e) crossing two knockout mice produced in step (d) to produce a homozygous alpha-4 integrin knockout mouse whose genome comprises two copies of the transgene;
- so that the knockout mouse of step (e) is unable to express functional alpha-4 integrin protein.
A method for assaying an agent for potential activity as an alpha-4 integrin protein antagonist, comprising the steps of:
- (a) administering the agent to a wild type mouse;
- (b) measuring the level of a genetic marker in the wild type mouse;
- and
- (c) comparing the measurement of step (b) with the level of the genetic marker measured in a control wild type mouse,
- wherein modulation of the level of the genetic marker measured in the wild type mouse relative to the level of the genetic marker measured in the control wild type mouse indicates the agent may possess alpha-4 integrin protein antagonist activity.
A method for assaying an agent for activity in ameliorating deleterious side effects associated with an alpha-4 integrin protein antagonist, comprising the steps of:
- (a) administering the agent to a mouse that is unable to express functional alpha-4 integrin;
- (b) measuring the level of a genetic marker in the mouse;
- and
- (c) comparing the level of the genetic marker measured in the mouse to the level of the genetic marker measured in a control mouse that is unable to express functional alpha 4 integrin,
- wherein a modulation of the level of the genetic marker measured in the mouse relative to the level of the genetic marker measured in the control mouse indicates the agent may have activity in ameliorating deleterious side effects associated with an alpha-4 integrin protein antagonist.
A method for assaying an agent for potential alpha-4 integrin protein antagonist activity, comprising the steps of:
- (a) removing a first blood sample from a mammal and measuring the concentration of progenitor stem cells in the first blood sample;
- (b) administering the agent to the mammal;
- (c) removing a second blood sample from the mammal and measuring the concentration of progenitor stem cells in the second blood sample;
- and
- (d) comparing the measured concentration of progenitor stem cells in the first blood sample with measured concentration of progenitor stem cells in the second blood sample,
- wherein an increase in the measured progenitor stem cell concentration in the second blood sample relative to the measured progenitor stem cell concentration in the first blood sample indicates the agent may have alpha-4 integrin protein antagonist activity.
A method for assaying an agent for potential alpha-4 integrin protein antagonist activity, comprising the steps of:
- (a) administering the agent to a mammal;
- (b) measuring the concentration of progenitor stem cells in the blood of the mammal;
- and
- (c) comparing the measured concentration of progenitor stem cells in the blood of the mammal to the measured concentration or progenitor stem cells in the blood of a control mammal,
- wherein an increase in the concentration of progenitor stem cells in the blood of the mammal relative to the concentration of progenitor stem cells in the blood of the control mammal is indicative of potential alpha-4 integrin protein antagonist activity in the agent.
A method for determining whether a compound or agent modulates signaling activity of a VLA-4 receptor, comprising the steps of:
- (a) administering the compound or agent to an organism;
- (b) measuring the expression level of a genetic marker for VLA-4 receptor signaling in a bodily sample removed from the organism;
- and
- (c) comparing the expression level of the genetic marker of step (b) with the expression level of the genetic marker measured in a control bodily sample,
- wherein a difference between the measured expression level of the genetic marker in the bodily sample and the control bodily sample indicates that the compound or agent modulates the signaling of the VLA-4 receptor.
A method for determining the efficacy of a potential antagonist of the signaling of a VLA-4 receptor, wherein such a method comprises the steps of:
- (a) removing a first bodily sample from an organism;
- (b) measuring the level of macrophage mannose receptor mRNA genetic marker in the first bodily sample;
- (c) administering the potential antagonist to the organism;
- (d) removing a second bodily sample from the organism;
- (e) measuring the level of the genetic marker macrophage mannose receptor mRNA in the second bodily sample;
- and
- (f) comparing the measured levels of step (b) and step (e),
- wherein a decrease in the level of the genetic marker measured in step (e) as compared to the level of the genetic marker measured in step (b) indicates the potential antagonist has efficacy in antagonizing the signaling activity of VLA-4.
A method for determining the ability of a compound or agent to modulate, and particularly to antagonize, the signaling activity of VLA-4 receptor, comprising the steps of:
- (a) contacting the compound or agent with a bodily sample from an organism;
- (b) measuring the expression level of a genetic marker for VLA-4 receptor signaling in the bodily sample;
- and
- (c) comparing the expression level of the genetic marker measured in step (b) with the expression level of the genetic marker measured in a control bodily sample.
A method for determining the efficacy of a potential antagonist of the signaling of a VLA-4 receptor, wherein such a method comprises the steps of:
- (a) removing a first bodily sample from a mouse;
- (b) measuring the level of Mus musculus mRNA for JAB, complete cds genetic marker in the first bodily sample;
- (c) administering the potential antagonist to the mouse;
- (d) removing a second bodily sample from the mouse;
- (e) measuring the level of the genetic marker Mus musculus mRNA for JAB, complete cds in the second bodily sample;
- and
- (f) comparing the measured levels of step (b) and step (e),
- wherein a decrease in the level of the genetic marker measured in step (e) as compared to the level of the genetic marker measured in step (b) indicates the potential antagonist has efficacy in antagonizing the signaling activity of VLA-4.
A method for determining the efficacy of a potential antagonist of the signaling of a VLA-4 receptor, wherein such a method comprises the steps of:
- (a) removing a first bodily sample from a mouse;
- (b) measuring the level of EST AA571535 genetic marker in the first bodily sample;
- (c) administering the potential antagonist to the mouse;
- (d) removing a second bodily sample from the mouse;
- (e) measuring the level of the genetic EST AA571535 in the second bodily sample;
- and
- (f) comparing the measured levels of step (b) and step (e),
- wherein a decrease in the level of the genetic marker measured in step (e) as compared to the level of the genetic marker measured in step (b) indicates the potential antagonist has efficacy in antagonizing the signaling activity of VLA-4.
Dependencies are inferred from the claim text.