The production of 4,182 mouse lines identifies experimental and biological variables impacting Cas9-mediated mutant mouse line production

AbstractThe International Mouse Phenotyping Consortium (IMPC) is generating and phenotyping null mutations for every protein-coding gene in the mouse1,2. The IMPC now uses Cas9, a programmable RNA-guided nuclease that has revolutionized mouse genome editing3 and increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of >3,300 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo null allele engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. Our analysis identified that whether a gene is essential for viability was the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for generating null alleles in mice using Cas9; these recommendations may be applicable to other allele types and species.

Download Full-text

Transmission of tauopathy strains is independent of their isoform composition

Nature Communications ◽

10.1038/s41467-019-13787-x ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 13

Author(s):

Zhuohao He ◽

Jennifer D. McBride ◽

Hong Xu ◽

Lakshmi Changolkar ◽

Soo-jung Kim ◽

...

Keyword(s):

Human Brain ◽

Transgenic Mouse ◽

Mouse Line ◽

Cell Type ◽

Transgenic Mouse Line ◽

Adult Human ◽

Tau Isoforms ◽

Underlying Mechanisms ◽

Mouse Lines

AbstractThe deposition of pathological tau is a common feature in several neurodegenerative tauopathies. Although equal ratios of tau isoforms with 3 (3R) and 4 (4R) microtubule-binding repeats are expressed in the adult human brain, the pathological tau from different tauopathies have distinct isoform compositions and cell type specificities. The underlying mechanisms of tauopathies are unknown, partially due to the lack of proper models. Here, we generate a new transgenic mouse line expressing equal ratios of 3R and 4R human tau isoforms (6hTau mice). Intracerebral injections of distinct human tauopathy brain-derived tau strains into 6hTau mice recapitulate the deposition of pathological tau with distinct tau isoform compositions and cell type specificities as in human tauopathies. Moreover, through in vivo propagation of these tau strains among different mouse lines, we demonstrate that the transmission of distinct tau strains is independent of strain isoform compositions, but instead intrinsic to unique pathological conformations.

Download Full-text

A resource of targeted mutant mouse lines for 5,061 genes

10.1101/844092 ◽

2019 ◽

Cited By ~ 4

Author(s):

Marie-Christine Birling ◽

Atsushi Yoshiki ◽

David J Adams ◽

Shinya Ayabe ◽

Arthur L Beaudet ◽

...

Keyword(s):

Mutant Mouse ◽

Embryonic Stem ◽

Null Alleles ◽

Mouse Mutant ◽

International Mouse Phenotyping Consortium ◽

Number Of Genes ◽

Mutant Strains ◽

Community Effort ◽

Mutant Mouse Line ◽

Public Repositories

AbstractThe International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background. This includes 2,850 null alleles for which no equivalent mutant mouse line exists, 2,987 novel conditional-ready alleles, and 4,433 novel reporter alleles. This nearly triples the number of genes with reporter alleles and almost doubles the number of conditional alleles available to the scientific community. When combined with more than 30 years of community effort, the total mutant allele mouse resource covers more than half of the genome. The extensively validated collection is archived and distributed through public repositories, facilitating availability to the worldwide biomedical research community, and expanding our understanding of gene function and human disease.

Download Full-text

Establishing F1A-CreERT2 Mice to Trace Fgf1 Expression in Adult Mouse Cardiomyocytes

Cells ◽

10.3390/cells11010121 ◽

2021 ◽

Vol 11 (1) ◽

pp. 121

Author(s):

Yi-Chao Hsu ◽

Yu-Fen Chung ◽

Mei-Shu Chen ◽

Chi-Kuang Wang ◽

Si-Tse Jiang ◽

...

Keyword(s):

Troponin T ◽

Lineage Tracing ◽

Mouse Line ◽

Protein Coding ◽

Physiological Processes ◽

Mrna Variants ◽

Alternatively Spliced

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo.

Download Full-text

Whole genome analysis for 163 guide RNAs in Cas9 edited mice reveals minimal off-target activity

10.1101/2021.08.11.455876 ◽

2021 ◽

Author(s):

Kevin A Peterson ◽

Sam Khalouei ◽

Joshua A Woodd ◽

Denise G Lanza ◽

Lauri G Lintott ◽

...

Keyword(s):

Inbred Strains ◽

Null Alleles ◽

Target Sequence ◽

Whole Genome ◽

Whole Genome Analysis ◽

Line Production ◽

Guide Rnas ◽

Bona Fide ◽

Target Activity

The Knockout Mouse Phenotyping Program (KOMP2) uses CRISRPR/Cas9 for high-throughput mouse line production to generate null alleles in the inbred C57BL/6N strain for broad-based in vivo phenotyping. In order to assess the risk of spurious S. pyogenes Cas9-induced off-target mutagenesis, we applied whole genome sequencing to compare the genomes of 50 Cas9-derived founder mice representing 163 different gRNAs to 28 untreated inbred control mice. Our analysis pipeline detected 28 off-target sequence variants associated with 21 guides. These potential off-targets were identified in 18/50 (36%) founders with 9/28 (32%) independently validated corresponding to 8 founder animals. In total, only 4.9% (8/163) of all guides exhibited off-target activity resulting in a rate of 0.16 Cas9 off-target mutations per founder analyzed. In comparison, we observed ~1225 unique variants in each mouse regardless of whether or not it was exposed to Cas9. These findings indicate that Cas9-mediated off-target mutagenesis is rare in founder knockout mice generated using guide RNAs designed to minimize off-target risk. Overall, bona fide off-target variants comprise a small fraction of the genetic heterogeneity found in carefully maintained colonies of inbred strains.

Download Full-text

Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

10.1101/195651 ◽

2017 ◽

Cited By ~ 1

Author(s):

Denise G. Lanza ◽

Angelina Gaspero ◽

Isabel Lorenzo ◽

Lan Liao ◽

Ping Zheng ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Gene Editing ◽

Null Allele ◽

Conditional Knockout ◽

Null Alleles ◽

Protein Coding ◽

International Mouse Phenotyping Consortium ◽

Conditional Allele ◽

In Cis

ABSTRACTThe International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-mediated homology driven repair (HDR) with short and long single-stranded oligodeoxynucleotides (ssODNs and lssODNs). Using pairs of guides and ssODNs donating loxP sites, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 of 30 genes targeted. LoxP sites integrated in cis in at least one F0 for 18 of 23 targeted genes. However, loxP sites were mutagenized in 4 of 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was not influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and a single lssODN to introduce a loxP-flanked exon, conditional allele founders were generated for all 4 genes targeted. Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssODNs are amenable to high-throughput production of conditional alleles when they can be employed.

Download Full-text

Unspecific expression in limited excitatory cell populations in interneuron-targeting Cre-driver lines can have large functional effects

10.1101/848655 ◽

2019 ◽

Author(s):

Daniel Müller-Komorowska ◽

Thoralf Opitz ◽

Shehabeldin Elzoheiry ◽

Michaela Schweizer ◽

Heinz Beck

Keyword(s):

Fluorescent Proteins ◽

Pyramidal Cells ◽

Cell Types ◽

Mouse Line ◽

Cell Type Specificity ◽

Functional Consequences ◽

Ca3 Pyramidal Cells ◽

Genetic Constructs ◽

Mouse Lines

1AbstractTransgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. Particularly, the investigation of interneurons has benefitted from the ability to target genetic constructs to defined cell types. However, the cell type specificity of some mouse lines has been called into questions. Here we show for the first time the functional consequences of unspecific expression in a somatostatin-Cre (SST-Cre) mouse line. We find large optogenetically evoked excitatory currents originating from unspecifically targeted CA3 pyramidal cells. We also used public Allen Brain Institute data to estimate expression specificity in other Cre lines. Another SST-Cre mouse lines shows comparable unspecificity, whereas a Parvalbumin-Cre mouse line shows much less unspecific expression. Finally, we make suggestions to ensure that the results from in-vivo use of Cre mouse lines are interpretable.

Download Full-text

Doubled haploid production in maize under Sub-montane Himalayan conditions using R1-nj-based haploid inducer TAILP1

Indian Journal of Genetics and Plant Breeding (The) ◽

10.31742/ijgpb.80.3.4 ◽

2020 ◽

Vol 80 (03) ◽

Author(s):

R. K Khulbe ◽

A. Pattanayak ◽

Lakshmi Kant ◽

G. S. Bisht ◽

M. C. Pant ◽

...

Keyword(s):

Doubled Haploid ◽

Sweet Corn ◽

Haploid Induction ◽

Maize Breeding ◽

Line Production ◽

Haploid Inducer ◽

Breeding Programmes ◽

Source Populations ◽

Developmental Phases

The use of in vivo haploid induction system makes the doubled haploid (DH) technology easier to adopt for the conventional maize breeders. However, despite having played an important role in the initial developmental phases of DH technology, Indian maize research has yet to harvest its benefits. Haploid Inducer Lines (HILs) developed by CIMMYT are being widely used in maize breeding programmes in many countries including India. There, however, is no published information on the efficiency of DH line production using CIMMYT HILs in Indian maize breeding programmes. In the present study, the efficiency of DH production using CIMMYT’s tropically adapted inducer line TAILP1 was investigated with eight source populations including two of sweet corn. The average haploid induction rate (HIR) of TAILP1 was 5.48% with a range of 2.01 to 10.03%. Efficiency of DH production ranged from 0.14 to 1.87% for different source populations with an average of 1.07%. The information generated will be useful for maize breeders intending to use DH technology for accelerated development of completely homozygous lines.

Download Full-text

Chimeric elk/mouse prion proteins in transgenic mice

Journal of General Virology ◽

10.1099/vir.0.045989-0 ◽

2013 ◽

Vol 94 (2) ◽

pp. 443-452 ◽

Cited By ~ 11

Author(s):

Gültekin Tamgüney ◽

Kurt Giles ◽

Abby Oehler ◽

Natrina L. Johnson ◽

Stephen J. DeArmond ◽

...

Keyword(s):

Neurodegenerative Disorder ◽

Prion Proteins ◽

Second Line ◽

Mouse Line ◽

Wasting Disease ◽

C Terminus ◽

Incubation Periods ◽

N Terminus ◽

Mouse Lines

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

Download Full-text

Fryl deficiency is associated with defective kidney development and function in mice

Experimental Biology and Medicine ◽

10.1177/1535370218758249 ◽

2018 ◽

Vol 243 (5) ◽

pp. 408-417 ◽

Cited By ~ 3

Author(s):

Yong-Sub Byun ◽

Eun-Kyoung Kim ◽

Kimi Araki ◽

Ken-ichi Yamamura ◽

Kihoon Lee ◽

...

Keyword(s):

Body Weight ◽

Growth Retardation ◽

Normal Development ◽

Full Term ◽

Lower Body ◽

Mouse Line ◽

Lower Body Weight ◽

Gene Functions ◽

Convoluted Tubules

FRY like transcription coactivator ( Fryl) gene located on chromosome 5 is a paralog of FRY microtubule binding protein ( Fry) in vertebrates. It encodes a protein with unknown functions. Fryl gene is conserved in various species ranging from eukaryotes to human. Although there are several reports on functions of Fry gene, functions of Fryl gene remain unclear. A mouse line containing null mutation in Fryl gene by gene trapping was produced in this study for the first time. The survival and growth of Fryl−/− mice were observed. Fryl gene expression levels in mouse tissues were determined and histopathologic analyses were conducted. Most Fryl−/− mice died soon after birth. Rare Fryl−/− survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl−/− survivors died of hydronephrosis before age 1. No abnormal histopathologic lesion was apparent in full-term embryo or adult tissues except the kidney. Abnormal lining cell layer detachments from walls of collecting and convoluted tubules in kidneys were apparent in Fryl−/− neonates and full-term embryos. Fryl gene was expressed in renal tubular tissues including the glomeruli and convoluted and collecting tubules. This indicates that defects in tubular systems are associated with Fryl functions and death of Fryl−/− neonates. Fryl protein is required for normal development and functional maintenance of kidney in mice. This is the first report of in vivo Fryl gene functions. Impact statement FRY like transcription coactivator ( Fryl) gene is conserved in various species ranging from eukaryotes to human. It expresses a protein with unknown function. We generated a Fryl gene mutant mouse line and found that most homozygous mice died soon after their birth. Rare Fryl−/− survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl−/− survivors died of hydronephrosis before age 1. Full-term mutant embryos showed abnormal collecting and convoluted tubules in kidneys where Fryl gene was expressed. Collectively, these results indicate that Fryl protein is required for normal development and functional maintenance of kidney in mice. To the best of our knowledge, this is the first report on in vivo Fryl gene functions.

Download Full-text