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Generation of Humanized Mouse Models with Focus on Antithrombin Deficiency

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Generation of Humanized Mouse Models with Focus on Antithrombin Deficiency. / Jensen, Astrid Bøgh.

2015. 119 p.

Research output: Book/anthology/dissertation/reportPh.D. thesisResearch

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@phdthesis{3ab9a6f9b90e4708a52fd0427557537a,
title = "Generation of Humanized Mouse Models with Focus on Antithrombin Deficiency",
abstract = "Antithrombin is one of the major inhibitors of the coagulation cascade. Antithrombin deficiency in humans significantly increases the risk of thrombosis. Mice homozygous for an antithrombin knock out die at day 16.5 of gestation, due to disseminated intravascular coagulation and organ failure. I found that homozygous knockout embryos have a significantly increased level of systemic inflammation, around their time of death. Furthermore, the embryos show signs of hypertension, already at day 12 of gestation, possible due to kidney failure. A humanized mouse model for antithrombin deficiency can be used to investigate human antithrombin mutations, or to test new therapeutics. I therefore attempted to partially humanize the murine coagulation system by expressing human antithrombin in mice homozygous for the antithrombin knock out allel. This transgene expression should rescue the lethal phenotype.Using pronucleus injection of a human antithrombin transgene with liver specific promoters I achieved a partial rescue of the lethal antithrombin knock out phenotype when the transgene was bred to homozygosity. No full rescue was obtained due to inadequate expression levels of the randomly integrated transgene. The CRISPR/Cas9 system is a relatively new and innovative method for targeted mutagenesis. The Cas9 nuclease introduces a double stranded break in the DNA, which can be repaired through homologous recombination of a targeting vector. A mutated Cas9n (Cas9 nickase) has been designed, which only cuts one of the DNA strands. With this enzyme, two target sites have to be located close to each other in order to create double strand break. This will lower the risk for off target mutations, but might reduce the efficiency of targeting.In order to control the expression of the human antithrombin gene by the murine antithrombin regulatory sequences, I designed a targeted mutagenesis using the CRISPR/Cas9 system which conserves the 5’UTR of the murine antithrombin gene. With the CRISPR/Cas9 I achieved targeting efficiency for heterozygous integrations of about 80{\%}, which correlated well with our preliminary CRISPR/Cas9 experiments targeting the Rosa26 locus. However, when targeting the Rosa26 locus, using the CRISPR/Cas9n system I only observed 65{\%} targeting efficiency for heterozygous integration which correlates well with the requirement for two nicks created by the mutated Cas9n.Others have shown that the CRISPR/Cas9 system can be directly used by a pronucleus injection to produce targeted knockout mice, thereby eliminate the time consuming ES cell work. Our preliminary results indicate that the CRISPR/Cas9n system can also be used directly in a pronucleus injection, to produce a targeted knockin mouse, despite its lower target efficiency",
author = "Jensen, {Astrid B{\o}gh}",
year = "2015",
month = "1",
day = "23",
language = "English",

}

RIS

TY - BOOK

T1 - Generation of Humanized Mouse Models with Focus on Antithrombin Deficiency

AU - Jensen, Astrid Bøgh

PY - 2015/1/23

Y1 - 2015/1/23

N2 - Antithrombin is one of the major inhibitors of the coagulation cascade. Antithrombin deficiency in humans significantly increases the risk of thrombosis. Mice homozygous for an antithrombin knock out die at day 16.5 of gestation, due to disseminated intravascular coagulation and organ failure. I found that homozygous knockout embryos have a significantly increased level of systemic inflammation, around their time of death. Furthermore, the embryos show signs of hypertension, already at day 12 of gestation, possible due to kidney failure. A humanized mouse model for antithrombin deficiency can be used to investigate human antithrombin mutations, or to test new therapeutics. I therefore attempted to partially humanize the murine coagulation system by expressing human antithrombin in mice homozygous for the antithrombin knock out allel. This transgene expression should rescue the lethal phenotype.Using pronucleus injection of a human antithrombin transgene with liver specific promoters I achieved a partial rescue of the lethal antithrombin knock out phenotype when the transgene was bred to homozygosity. No full rescue was obtained due to inadequate expression levels of the randomly integrated transgene. The CRISPR/Cas9 system is a relatively new and innovative method for targeted mutagenesis. The Cas9 nuclease introduces a double stranded break in the DNA, which can be repaired through homologous recombination of a targeting vector. A mutated Cas9n (Cas9 nickase) has been designed, which only cuts one of the DNA strands. With this enzyme, two target sites have to be located close to each other in order to create double strand break. This will lower the risk for off target mutations, but might reduce the efficiency of targeting.In order to control the expression of the human antithrombin gene by the murine antithrombin regulatory sequences, I designed a targeted mutagenesis using the CRISPR/Cas9 system which conserves the 5’UTR of the murine antithrombin gene. With the CRISPR/Cas9 I achieved targeting efficiency for heterozygous integrations of about 80%, which correlated well with our preliminary CRISPR/Cas9 experiments targeting the Rosa26 locus. However, when targeting the Rosa26 locus, using the CRISPR/Cas9n system I only observed 65% targeting efficiency for heterozygous integration which correlates well with the requirement for two nicks created by the mutated Cas9n.Others have shown that the CRISPR/Cas9 system can be directly used by a pronucleus injection to produce targeted knockout mice, thereby eliminate the time consuming ES cell work. Our preliminary results indicate that the CRISPR/Cas9n system can also be used directly in a pronucleus injection, to produce a targeted knockin mouse, despite its lower target efficiency

AB - Antithrombin is one of the major inhibitors of the coagulation cascade. Antithrombin deficiency in humans significantly increases the risk of thrombosis. Mice homozygous for an antithrombin knock out die at day 16.5 of gestation, due to disseminated intravascular coagulation and organ failure. I found that homozygous knockout embryos have a significantly increased level of systemic inflammation, around their time of death. Furthermore, the embryos show signs of hypertension, already at day 12 of gestation, possible due to kidney failure. A humanized mouse model for antithrombin deficiency can be used to investigate human antithrombin mutations, or to test new therapeutics. I therefore attempted to partially humanize the murine coagulation system by expressing human antithrombin in mice homozygous for the antithrombin knock out allel. This transgene expression should rescue the lethal phenotype.Using pronucleus injection of a human antithrombin transgene with liver specific promoters I achieved a partial rescue of the lethal antithrombin knock out phenotype when the transgene was bred to homozygosity. No full rescue was obtained due to inadequate expression levels of the randomly integrated transgene. The CRISPR/Cas9 system is a relatively new and innovative method for targeted mutagenesis. The Cas9 nuclease introduces a double stranded break in the DNA, which can be repaired through homologous recombination of a targeting vector. A mutated Cas9n (Cas9 nickase) has been designed, which only cuts one of the DNA strands. With this enzyme, two target sites have to be located close to each other in order to create double strand break. This will lower the risk for off target mutations, but might reduce the efficiency of targeting.In order to control the expression of the human antithrombin gene by the murine antithrombin regulatory sequences, I designed a targeted mutagenesis using the CRISPR/Cas9 system which conserves the 5’UTR of the murine antithrombin gene. With the CRISPR/Cas9 I achieved targeting efficiency for heterozygous integrations of about 80%, which correlated well with our preliminary CRISPR/Cas9 experiments targeting the Rosa26 locus. However, when targeting the Rosa26 locus, using the CRISPR/Cas9n system I only observed 65% targeting efficiency for heterozygous integration which correlates well with the requirement for two nicks created by the mutated Cas9n.Others have shown that the CRISPR/Cas9 system can be directly used by a pronucleus injection to produce targeted knockout mice, thereby eliminate the time consuming ES cell work. Our preliminary results indicate that the CRISPR/Cas9n system can also be used directly in a pronucleus injection, to produce a targeted knockin mouse, despite its lower target efficiency

M3 - Ph.D. thesis

BT - Generation of Humanized Mouse Models with Focus on Antithrombin Deficiency

ER -