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The Department of Molecular Mechanisms of Ontogenesis actively uses novel methods to generate mice with targeted genome modifications. We successfully collaborate with investigators from Institute Cytology and Genetics (Russia, Novosibirsk) and other Russian institutes.

To generate genetically modified mice, we use two main strategies: direct modification of the zygote genome or the chimeras production. With these two approaches we create transgenic mice with random transgene integrations or mice with targeted modifications (using the CRISPR/Cas system).

Over the past 5 years, more than 20 different lines with various genome modifications have been successfully obtained in our department. At the moment, we have extensive experience in the field of mouse genome modification.

We invite scientific groups for cooperation in the transgenic mice production. Our department has a full cycle for producing genetically-modified mice: from design of targeted modifications and genetic constructs to the generation of genome-modified mouse lines.

 

Our competences:

  • Design of experiment including CRISPR and genetic constructs;
  • Generation of transgenic mice with random integration sites by traditional pronuclear microinjection;
  • Generation of knockout mice;
  • Generation of mice with targeted point mutations;
  • Generation of knock-in mice;
  • Generation of humanized mice;
  • Generation of mice with chromosomal aberrations.

 

For additional information please contact:

Nariman Battulin

e-mail:

battulin@gmail.com

battulin@bionet.nsc.ru

Work phone:

+7 (383) 363-49-63*1110

 

Mouse lines

 

1)      C57BL/6J-Panx1em1Koral/Icg – knockout of Panx1 gene by deleting coding exons 3 and 4 

This line was generated in collaboration with Panchin Yuri from Institute for information transmission problems, Russia, Moscow (http://iitp.ru/ru/researchlabs/241.htm). The mouse line was donated in SPF-vivarium of Institute of Cytology and Genetics, Russia, Novosibirsk (http://spf.bionet.nsc.ru/).

The knockout mouse line was generated by deletion of coding exons 3 and 4 by microinjection of CRISPR/Cas9 components into fertilized mouse oocytes of C57BL/6J inbred strain.

Genetics:

  • Deletion of coding exons 3 and 4 of Panx1 gene;
  • Deletion size — 3,473 bp;
  • Genomic coordinates — chr9: 15006884-15010356 (mm10).

References:

 

2)      C57BL/6J-Panx1em2Koral/Icg – humanization of Panx1 gene by codon substitution R216H

This line was generated in collaboration with Panchin Yuri from Institute for information transmission problems, Russia, Moscow (http://iitp.ru/ru/researchlabs/241.htm). After, the mouse line was donated in SPF-vivarium of Institute of Cytology and Genetics, Russia, Novosibirsk (http://spf.bionet.nsc.ru/).

The mouse line was generated by HDR-mediated substitution of arginine to histidine in the  position 216 of Panx1 gene. Fertilized mouse oocytes of C57BL/6J inbred strain were injected with a mix of ssODN, Cas9-mRNA and gRNA. This line mimicked human R217H mutation.

Genetics:

  • Codon substitution R216H (CGG to CAC) in the Panx1 gene.

References:

 

3)      C57BL/6J-H2-Ab1em1Koral/Icg – knockout of H2-Ab1 gene by whole gene deletion

The knockout mouse line was generated by whole gene deletion using microinjection of CRISPR components into mouse zygotes.

Homozygotes for targeted null mutations exhibit depletion of mature CD4+ T cells, deficiency in cell-mediated immune responses, and increased susceptibility to viral infections.

This mouse line was donated in SPF-vivarium of Institute of Cytology and Genetics, Russia, Novosibirsk (http://spf.bionet.nsc.ru/).

Two independent mouse lines, C57BL/6J-H2-Ab1em2Koral/Icg and C57BL/6J-H2-Ab1em3Koral/Icg, have similar genetic characteristics.

Genetics:

  • Full deletion of H2-Ab1 gene;
  • Deletion size — 5.8 kb;
  • Genome coordinates — hr17: 34263304-34269093 (mm10).

References:

 

4)      C57BL/6J-Pcbp1em1Koral/Icg – knockout of Pcbp1 gene 

This line was generated in collaboration with Alexey Tomilin from Institute of Cytology, Saint-Petersburg, Russia, (https://www.incras.ru/institut/nauchnye-podrazdelenija/laboratorija-molekuljarnoj-biologii-stvolovyh-kletok/). This line is stored in cryoconservation status in our department.

Mice heterozygous for a knock-out allele exhibit reduced body weight. Mice homozygous for the allele exhibit embryonic lethality between E3.5 and E8.5.

Genetics:

  • 47 bp deletion including the ATG codon in the first exon of Pcbp1 gene
  • Genomic coordinates — chr6: 86525884-86525930 (mm10)

References:

 

 5)      C57BL/6J-Pcbp1em2Koral/Icg –  knockout of Pcbp1 gene

This line was generated in collaboration with Alexey Tomilin from Institute of Cytology,  Saint-Petersburg, Russia,(https://www.incras.ru/institut/nauchnye-podrazdelenija/laboratorija-molekuljarnoj-biologii-stvolovyh-kletok/). This line is stored in cryoconservation status in our department.

Mice heterozygous for a knock-out allele exhibit reduced body weight. Mice homozygous for the allele exhibit embryonic lethality between E3.5 and E8.5.

Genetics:

  • 38 bp insertion in first exon after the 4th codon of Pcbp1 gene;
  • Genomic coordinates — chr6: 86525990 (mm10).

References:

 

6)      C57BL/6J-Cntn6em1Koral/Icg – 1.137 Mb deletion of Cntn6 gene 

This line was generated by full-length gene deletion including 3’ and 5’ regulatory regions. To generate targeted deletion, zygotes from C57BL/6J inbred strain were injected with a mix of Cas9-mRNA, two gRNA and ssODN.

This mouse line was donated in SPF-vivarium of Institute of Cytology and Genetics, Russia, Novosibirsk (http://spf.bionet.nsc.ru/).

Genetics:

  • Full gene deletion including 3’ and 5’ regulatory regions;
  • Deletion size — 1.137 Mb;
  • Genomic coordinates — chr6: 103842569-104979807 (mm10).

Six independent mouse lines have similar genetic characteristics and are stored in the cryo-archive of our department.

References:

  1. Korablev, A.N., Serova, I.A. & Serov, O.L. Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology. BMC Genet 18, 112 (2017). https://doi.org/10.1186/s12863-017-0582-7
  2. Pristyazhnyuk, I.E., Minina, J., Korablev, A. et al. Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice. Sci Rep 9, 14161 (2019). https://doi.org/10.1038/s41598-019-50649-4

 

7)      B6.Cg-Cntn6em2Koral/Icg – 1.137 Mb duplication of Cntn6 gene 

This line was generated by duplication of Cntn6 gene including 3’ and 5’ regulatory regions. To generate duplication, zygotes from C57BL/6J x CBA hybrid strain were injected with a mix of CRISPR components. Then, each mouse line was backcrossed on C57BL/6J inbred strain for more than 5 generations. As a result, we obtained two independent mouse lines.

Genetics:

  • Duplication of Cntn6 gene including 3’ and 5’ regulatory regions;
  • Size of duplication — 1.137 Mb;
  • Genomic coordinates chr6: 103842569-104979807 (mm10).

One independent mouse line has similar genetic characteristics and is stored in the cryo archive of our department.

References:

  1. Korablev, A.N., Serova, I.A. & Serov, O.L. Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology. BMC Genet 18, 112 (2017). https://doi.org/10.1186/s12863-017-0582-7
  2. Pristyazhnyuk, I.E., Minina, J., Korablev, A. et al. Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice. Sci Rep 9, 14161 (2019). https://doi.org/10.1038/s41598-019-50649-4

 

8)  B6.Cg-Cntn6em3Koral/Icg – 1.137 Mb inversion of Cntn6 gene 

This line was generated by full-length gene inversion including 3’ and 5’ regulatory regions. To generate inversion, zygotes from C57BL/6J x CBA hybrid strain were injected with a mix of CRISPR components. Then, each mouse line was backcrossed on C57BL/6J inbred strain for more than 5 generations. As a result, we obtained two independent mouse lines.

Genetics:

  • Inversion of Cntn6 including 3’ and 5’ regulatory regions;
  • Size of inversion — 1.137 Mb;
  • Genomic coordinates — chr6: 103842569-104979807 (mm10).

One independent mouse line has similar genetic characteristics and is stored in the cryo-archive of our department.

References:

  1. Korablev, A.N., Serova, I.A. & Serov, O.L. Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology. BMC Genet 18, 112 (2017). https://doi.org/10.1186/s12863-017-0582-7

 

 

9)  C57BL/6J-Tnfem1Koral/Icg – knockout of Tnf-α gene

The knockout mouse line was generated by a 16 bp insertion, introducing a frameshift mutation in the first exon. To generate knockout mouse strain, zygotes from C57BL/6J inbred strain were injected with a mix of Cas9-protein, gRNA and ssODN. This line was generated in collaboration with  Darya Bazovkina from Institute of Cytology and Genetics, Russia, Novosibirsk.

Genetics:

  • 16 bp insertion in the first exon, leading to nonsense frameshift mutation.

References:

 

12)  C57BL/6J-Ptpn5em1Koral/Icg – knockout of Ptpn5 gene

The knockout mouse line was generated by microinjection of CRISPR components into C57BL/6J mouse zygotes leading to deletion of the 12-13 exons. This line was generated in collaboration with Elizaveta Kulikova from Institute of Cytology and Genetics, Russia, Novosibirsk.

Genetics:

  • Deletion of exon 12 (partial) and exon 13 (full), which affected Ptpn5 substrate-binding domain;
  • Deletion size — 347 bp;
  • Genome coordinates — chr7: 47079194-47079540 (mm10).

References:

 

10)  B6.Cg-Kitem1Koral/Icg — knockout of the Kit gene including deletion of a TAD border

This line was generated by microinjection of CRISPR components into mouse zygotes. Spontaneous deletion was formed at the site of CRISPR-induced DSB.

To generate modification, zygotes from C57BL/6J x CBA hybrid strain were injected with a mix of CRISPR components. Each mouse line was backcrossed on C57BL/6J inbred strain for more than 5 generations.

Genetics:

  • Deletion of exons 2-21 of the Kit gene including the TAD border between the Kit locus and the Kdr locus. Only the first exon coding for the KIT signal peptide remained intact (aa sequence MRGARGAWDLLCVLLVLLRGQT);
  • Deletion size — 293 kb;
  • Genomic coordinates — chr5: 75588218-75881214 (mm10).

Phenotype:

  • Mutations at this locus affect migration of embryonic stem cell populations, resulting in mild to severe impairments in hematopoiesis and pigmentation. While wild-type mice have black coats, heterozygous mutants demonstrate additional abdominal white spots.

References:

  1. Korablev, A.; Lukyanchikova, V.; Serova, I.; Battulin, N. On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes. J. Mol. Sci. 2020, 21, 3604. https://doi.org/10.3390/ijms21103604

 

11) B6;129P-Csn1s1tm1Icg/Icg – insertion of the human GMCSF gene after the promoter of the endogenous mouse Csn1s1 (α-S1-casein) gene

The mouse line was created by injections of transgenic 129S2/SvPasCrl mESCs into blastocysts resulting in chimeric animals. Experiments were performed by Dr. Galina Kontsevaya at SPF-vivarium of Institute of Cytology and Genetics, Novosibirsk, Russia.

Genetics:

  • Insertion of the human GMCSF gene (2029 bp) into the mouse Csn1s1 gene ORF, right after the start codon.
  • Genomic coordinates — chr5: 87667246 (mm10).

Phenotype:

  • Pups from homozygous females grow slower due to the lack of α-S1-casein in the milk of lactating females.
  • Milk from transgenic females contains 2-5 ug/ml hGMCSF.

 

 12) B6;129P-Csn1s1tm2Icg/Icg – replacement of the mouse Csn1s1 (α-S1-casein) gene with the human GMCSF gene

The mouse line was created by injections of transgenic 129S2/SvPasCrl mESCs into blastocysts resulting in chimeric animals. Experiments were performed by Dr. Galina Kontsevaya at SPF-vivarium of Institute of Cytology and Genetics, Novosibirsk, Russia.

enetics:

  • Insertion of the human GMCSF gene (2029 bp) into the mouse Csn1s1 gene ORF, right after the start codon. Concomitantly, the Csn1s1 coding region (13,7 kbp) was deleted.
  • Genomic coordinates — chr5: 87667246-87680963 (mm10).

Phenotype:

  • Pups from homozygous females grow slower due to the lack of α-S1-casein in the milk of lactating females.
  • Milk from transgenic females contains 100-500 ug/ml hGMCSF.