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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

Germ line transmission and establishment of a homozygous Cr4 insertion mutation. (A) A sequencing reaction chromatogram from a founder mouse showing a correct donor-oligo insertion at the Cr4 target site. (B) The sequence confirmed mutant was bred with a wild type mouse and germ line transmission was established. Genotyping of F2 generation litter with flanking primers PCR (left) and with Internal + External primers PCR (right). The expected product sizes and the genotypes are given below the gel images. M: 100 base pair Marker. (C) Nucleotide sequence of the locus containing the correct insertion sequence. The locations of primers are shown with the corresponding color coded arrows. Cr4 sequence is underlined and PAM sequence is shown in uppercase.
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f0015: Germ line transmission and establishment of a homozygous Cr4 insertion mutation. (A) A sequencing reaction chromatogram from a founder mouse showing a correct donor-oligo insertion at the Cr4 target site. (B) The sequence confirmed mutant was bred with a wild type mouse and germ line transmission was established. Genotyping of F2 generation litter with flanking primers PCR (left) and with Internal + External primers PCR (right). The expected product sizes and the genotypes are given below the gel images. M: 100 base pair Marker. (C) Nucleotide sequence of the locus containing the correct insertion sequence. The locations of primers are shown with the corresponding color coded arrows. Cr4 sequence is underlined and PAM sequence is shown in uppercase.

Mentions: Higher migrating sharper bands from flanking PCR assay for a few samples were purified and sequenced. While some samples showed clean sequence (Fig. 3A) with precise insertion of the donor-oligo sequence, others contained mixtures of sequences indicative of mosaicism (data not shown), possibly due to insertions occurring after the 2 cell stage [4]. Since we obtained more than sufficient number of founders with the precise oligo sequence insertion, we only bred two founders each (for Cr2 or Cr4 insertions) from these and we did not pursue breeding of the other founders. Out of curiosity, we further analyzed the nature of mutations in a few samples that showed atypical banding pattern in the genotyping PCRs. The results uncovered some anomalous mutations in those samples that are presented below.


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)

Germ line transmission and establishment of a homozygous Cr4 insertion mutation. (A) A sequencing reaction chromatogram from a founder mouse showing a correct donor-oligo insertion at the Cr4 target site. (B) The sequence confirmed mutant was bred with a wild type mouse and germ line transmission was established. Genotyping of F2 generation litter with flanking primers PCR (left) and with Internal + External primers PCR (right). The expected product sizes and the genotypes are given below the gel images. M: 100 base pair Marker. (C) Nucleotide sequence of the locus containing the correct insertion sequence. The locations of primers are shown with the corresponding color coded arrows. Cr4 sequence is underlined and PAM sequence is shown in uppercase.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: Germ line transmission and establishment of a homozygous Cr4 insertion mutation. (A) A sequencing reaction chromatogram from a founder mouse showing a correct donor-oligo insertion at the Cr4 target site. (B) The sequence confirmed mutant was bred with a wild type mouse and germ line transmission was established. Genotyping of F2 generation litter with flanking primers PCR (left) and with Internal + External primers PCR (right). The expected product sizes and the genotypes are given below the gel images. M: 100 base pair Marker. (C) Nucleotide sequence of the locus containing the correct insertion sequence. The locations of primers are shown with the corresponding color coded arrows. Cr4 sequence is underlined and PAM sequence is shown in uppercase.
Mentions: Higher migrating sharper bands from flanking PCR assay for a few samples were purified and sequenced. While some samples showed clean sequence (Fig. 3A) with precise insertion of the donor-oligo sequence, others contained mixtures of sequences indicative of mosaicism (data not shown), possibly due to insertions occurring after the 2 cell stage [4]. Since we obtained more than sufficient number of founders with the precise oligo sequence insertion, we only bred two founders each (for Cr2 or Cr4 insertions) from these and we did not pursue breeding of the other founders. Out of curiosity, we further analyzed the nature of mutations in a few samples that showed atypical banding pattern in the genotyping PCRs. The results uncovered some anomalous mutations in those samples that are presented below.

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