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Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system.

Quadros RM, Harms DW, Ohtsuka M, Gurumurthy CB - FEBS Open Bio (2015)

Bottom Line: One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site.We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools.This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

View Article: PubMed Central - PubMed

Affiliation: Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based Targeted Transgenesis (PITT), a method that uses a recombinase-mediated cassette exchange (RMCE) to enable insertion of sequences at a predetermined genomic locus, such as ROSA26. The PITT technique uses fertilized eggs (instead of ES cells) collected from 'seed mice' that contain the RMCE landing pad. The PITT method can rapidly generate reliable targeted transgenic mice; it requires a seed mouse, which in our previous study was generated using ES cell targeting approaches. Here, we demonstrate that seed mice containing the RMCE landing pad can be developed rapidly by using the CRISPR/Cas9 system. One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site. We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools. This two-step strategy combines the best features of the two newer technologies-rapid creation of PITT landing pads using the CRISPR/Cas9 system and efficient and precise insertion of larger cassettes at the landing pads using PITT. This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

No MeSH data available.


Related in: MedlinePlus

Genotyping of offspring. (A) Schematic of Flanking Primer PCR assay. The primers bind on the flanking sequences. Insertion of the JT15-Spacer-Lox2272 sites adds 78 bases at the target site that increases the PCR band size from 419 bp (wild type) to 497 bp (mutant). Some samples show incorrect sized mutant band (see text for details). (B) Internal + External primer PCR. Genotyping PCR with a forward primer in the repair oligo region and a reverse primer downstream of Cr4 site. Insertion at Cr2 and Cr4 sites will yield bands of 410 and 225 base pairs respectively. See text for discussion on results for samples 6 and 53. M: 100 base pair Marker.
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f0010: Genotyping of offspring. (A) Schematic of Flanking Primer PCR assay. The primers bind on the flanking sequences. Insertion of the JT15-Spacer-Lox2272 sites adds 78 bases at the target site that increases the PCR band size from 419 bp (wild type) to 497 bp (mutant). Some samples show incorrect sized mutant band (see text for details). (B) Internal + External primer PCR. Genotyping PCR with a forward primer in the repair oligo region and a reverse primer downstream of Cr4 site. Insertion at Cr2 and Cr4 sites will yield bands of 410 and 225 base pairs respectively. See text for discussion on results for samples 6 and 53. M: 100 base pair Marker.

Mentions: The target sequences from the genomic DNA of offspring were amplified using flanking primers and analyzed in a 2% agarose gel. As expected, some samples had slower migrating bands indicative of donor oligonucleotide insertion (Fig. 2A). While there were many founders that had the expected increase in size, some (founders 6, 9, 14, 15, 16, 20 and 23) had higher than the expected sized bands (Fig. 2A). The higher sized (or incorrect sized bands) may be the result of scenarios such as (i) addition of more than one copy of the donor-oligo, or (ii) duplication of some adjacent sequences near the target site before the donor-oligo gets inserted, or (iii) combination of varying lengths of deletion & addition of nucleotides followed by insertion of the donor-oligo, or (iv) due to unequal amplification of templates (higher sized bands being less favored) in the PCR reaction.


Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system.

Quadros RM, Harms DW, Ohtsuka M, Gurumurthy CB - FEBS Open Bio (2015)

Genotyping of offspring. (A) Schematic of Flanking Primer PCR assay. The primers bind on the flanking sequences. Insertion of the JT15-Spacer-Lox2272 sites adds 78 bases at the target site that increases the PCR band size from 419 bp (wild type) to 497 bp (mutant). Some samples show incorrect sized mutant band (see text for details). (B) Internal + External primer PCR. Genotyping PCR with a forward primer in the repair oligo region and a reverse primer downstream of Cr4 site. Insertion at Cr2 and Cr4 sites will yield bands of 410 and 225 base pairs respectively. See text for discussion on results for samples 6 and 53. M: 100 base pair Marker.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372609&req=5

f0010: Genotyping of offspring. (A) Schematic of Flanking Primer PCR assay. The primers bind on the flanking sequences. Insertion of the JT15-Spacer-Lox2272 sites adds 78 bases at the target site that increases the PCR band size from 419 bp (wild type) to 497 bp (mutant). Some samples show incorrect sized mutant band (see text for details). (B) Internal + External primer PCR. Genotyping PCR with a forward primer in the repair oligo region and a reverse primer downstream of Cr4 site. Insertion at Cr2 and Cr4 sites will yield bands of 410 and 225 base pairs respectively. See text for discussion on results for samples 6 and 53. M: 100 base pair Marker.
Mentions: The target sequences from the genomic DNA of offspring were amplified using flanking primers and analyzed in a 2% agarose gel. As expected, some samples had slower migrating bands indicative of donor oligonucleotide insertion (Fig. 2A). While there were many founders that had the expected increase in size, some (founders 6, 9, 14, 15, 16, 20 and 23) had higher than the expected sized bands (Fig. 2A). The higher sized (or incorrect sized bands) may be the result of scenarios such as (i) addition of more than one copy of the donor-oligo, or (ii) duplication of some adjacent sequences near the target site before the donor-oligo gets inserted, or (iii) combination of varying lengths of deletion & addition of nucleotides followed by insertion of the donor-oligo, or (iv) due to unequal amplification of templates (higher sized bands being less favored) in the PCR reaction.

Bottom Line: One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site.We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools.This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

View Article: PubMed Central - PubMed

Affiliation: Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based Targeted Transgenesis (PITT), a method that uses a recombinase-mediated cassette exchange (RMCE) to enable insertion of sequences at a predetermined genomic locus, such as ROSA26. The PITT technique uses fertilized eggs (instead of ES cells) collected from 'seed mice' that contain the RMCE landing pad. The PITT method can rapidly generate reliable targeted transgenic mice; it requires a seed mouse, which in our previous study was generated using ES cell targeting approaches. Here, we demonstrate that seed mice containing the RMCE landing pad can be developed rapidly by using the CRISPR/Cas9 system. One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site. We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools. This two-step strategy combines the best features of the two newer technologies-rapid creation of PITT landing pads using the CRISPR/Cas9 system and efficient and precise insertion of larger cassettes at the landing pads using PITT. This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

No MeSH data available.


Related in: MedlinePlus