FVB/N-Tg(Prnp-PFN1*C71G)22Zxu Tg(Thy1-PFN1*C71G)67Zxu/J

Stock number: 028608
Product name: Thy1.2-PFN1^C71G/C71G/Prp-PFN1^C71G triple transgenic [or pt(h)] , Thy1.2-PFN1^C71G/Prp-PFN1^C71G double transgenic [or pt]
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Thy1.2-PFN1^C71G/Prp-PFN1^C71G transgenic mice ("pt" or "pt(h)") express a C71G mutant human profilin 1 protein (with N-terminal V5 tag) predominantly in motor neurons and in both neurons and non-neuronal cells of the CNS. These mice exhibit motor neuron degeneration and abnormalities in cytoskeleton, proteasome, autophagy and stress granules. This is a model for studying the pathogenic role of PFN1 in familial amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease).

Detailed description

These mice harbor two transgenes, Prp-PFN1^C71G and Thy1.2-PFN1^C71G, each expressing a C71G mutant human profilin 1 protein with N-terminal V5 tag (V5-PFN1^C71G) under direction of the mouse prion protein promoter or the human Thy1.2 promoter.

The Prp-PFN1^C71G transgene has V5-PFN1^C71G expression predominantly in the CNS (in both neurons and non-neuronal cells), with very low expression in the periphery. Specifically, expression is highest in the spinal cord, brainstem and forebrain, lower levels in cerebellum and kidney, and at undetectable levels in other organs.

The Thy1.2-PFN1^C71G transgene drives V5-PFN1^C71G expression predominantly in motor neurons. Specifically, expression is highest in the spinal cord and brainstem, followed by the forebrain. Little if any is expressed in the cerebellum and the peripheral organs.

When hemizygous for both transgenes, they are referred to as Thy1.2-PFN1^C71G/Prp-PFN1^C71G double transgenic mice (and were previously called "pt" mice).
When homozygous for Thy1.2-PFN1^C71G and hemizygous for Prp-PFN1^C71G are referred to as Thy1.2-PFN1^C71G/C71G/Prp-PFN1^C71G triple transgenic mice (and were previously called "pt(h)" mice).
Furthermore, mice harboring only the Thy1.2-PFN1^C71G transgene are referred to as Thy1.2-PFN1^C71G (previously called "t") when hemizygous, and Thy1.2-PFN1^C71G/C71G (previously called "t(h)") when homozygous.
Similarly, mice harboring only the Prp-PFN1^C71G transgene are referred to as Prp-PFN1^C71G (previously called "p") when hemizygous, and Prp-PFN1^C71G/C71G (previously called "p(h)") when homozygous.

In order of most-to-least motor neuron disease severity: "pt(h)" : "t(h)" : "pt" : "t" : "p".

The "pt(h)" and "t(h)" mice have similar disease onset age (~125d), but "pt(h)" mice develop paralysis faster than "t(h)" mice (~60d after onset compared to ~170d after onset). While "t(h)" and "pt" mice have similar overall life expectancy (~250-350d), the "pt" mice have a much later disease onset (~250d) with rapid development of paralysis (~10d after onset).

The "pt(h)" genotype is most severely affected, with mice exhibiting motor neuron degeneration and abnormalities in cytoskeleton, proteasome, autophagy and stress granules. Specifically, beginning at 3-4 months of age, "pt(h)" exhibit progressive loss of motor capabilities, with progressive loss of motor and sensory axons. Cytoskeleton defects in motor neurons include mutant PFN1 aggregation and reduced neurofilament L expression. Alterations in autophagy pathway are evident throughout spinal cord, particularly in motor neurons. At the paralysis disease stage (end stage), there is significant microgliosis, astrogliosis, motor neuron loss, elevated ubiquitin levels in the spinal cord, increased stress granules in the spinal motor neurons

Both "pt" and "pt(h)" mice are viable and fertile. The donating investigator reports that "pt(h)" mice breed normally, and they maintain their double transgenic colony by breeding "t(h)" females with "pt(h)" males.

Mice singly hemizygous for the Prp-PFN1^C71G transgene ("p") express V5-PFN1^C71G in both neurons and glia, but the expression levels are low. The "p" genotype does not manifest motor neuron degeneration symptoms during their lifespan. The donating investigator also reports they were not able to obtain homozygous Prp-PFN1^C71G animals ("p(h)").

Development

These mice harbor two transgenes, Prp-PFN1^C71G and Thy1.2-PFN1^C71G, each designed by Drs. Zuoshang Xu and John E. Landers (University of Massachusetts Medical School). The PFN1^C71G protein (V5-PFN1^C71G) encoded by each transgene is a human profilin 1 cDNA sequence (PFN1; 423 bp / 141 aa) harboring the C71G mutation associated with amyotrophic lateral sclerosis (ALS), with a V5-tag (45 bp) fused to its N-terminus.

The Prp-PFN1^C71G transgene has the V5-PFN1^C71G sequence inserted between exon 2 and exon 3 of mouse prion protein (PrP or Prnp) gene at a unique XhoI site in the MoPrP.Xho plasmid vector. The resulting 11,855 bp Prp-V5-PFN1 construct was microinjected into FVB/NJ embryos. Transgenic founder male 22 was bred with FVB/NJ females for germline transmission, and founder line 22 was found to have expression predominantly in the CNS. The integration site(s) and copy number of the Prp-PFN1^C71G transgene is not known to date (February 2016). The Prp-PFN1^C71G line 22 colony was maintained by breeding hemizygotes with FVB/NJ mice for at least one more generation, prior to using as below.

The Thy1.2-PFN1^C71G transgene has the V5-PFN1^C71G sequence inserted downstream of the modified ~6.5 kbp mouse Thy1.2 promoter sequences (Thy1; thymus cell antigen 1, theta). The resulting 1,171 bp Thy1-V5-PFN1 construct was microinjected into FVB/NJ embryos. Transgenic founder female 67 was bred with FVB/NJ males for germline transmission, and founder line 67 was found to have expression predominantly in the CNS. The integration site(s) and copy number of the Thy1.2-PFN1^C71G transgene is not known to date (February 2016). The Thy1.2-PFN1^C71G line 67 colony was maintained by breeding hemizygotes with FVB/NJ mice for at least one more generation, prior to using as below.

To generate the Prp-PFN1^C71G Thy1.2-PFN1^C71G mice, the two individual mouse lines were bred together. The donating investigator then maintained the colony by breeding females homozygous for Thy1.2-PFN1^C71G ("t(h)" females) with males hemizygous for PrP-PFN1^C71G and homozygous for Thy1.2-PFN1^C71G ("pt(h)" males). In 2016, albino males harboring both transgenes were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To generate our live colony, an aliquot of the frozen sperm was used to fertilize oocytes from FVB/NJ mice (Stock No. 001800).

Allele

Symbol: Tg(Prnp-PFN1*C71G)22Zxu
Name: transgene insertion 22, Zuoshang Xu
MGI accession ID: MGI:5796267
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(Prnp-PFN1*C71G)22Zxu
Marker name: transgene insertion 22, Zuoshang Xu
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: PFN1,profilin 1,human
Molecular note: The transgenic construct contains a mutant human profilin 1 cDNA sequence with a V5 tag (45 bp) fused to the N-terminus and inserted between exon 2 and exon 3 of mouse prion protein gene at a unique XhoI site in the MoPrP.Xho plasmid vector. PFN1 harbors the C71G mutation associated with amyotrophic lateral sclerosis (ALS).

Allele

Symbol: Tg(Thy1-PFN1*C71G)67Zxu
Name: transgene insertion 67, Zuoshang Xu
MGI accession ID: MGI:5796269
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(Thy1-PFN1*C71G)67Zxu
Marker name: transgene insertion 67, Zuoshang Xu
Chromosome: UN
Strain of origin: Not Specified
Expressed genes: PFN1,profilin 1,human
Molecular note: The transgenic construct contains a mutant human profilin 1 cDNA sequence with a V5 tag (45 bp) fused to the N-terminus and inserted downstream of the modified ~6.5 kbp mouse Thy1.2 promoter sequences. PFN1 harbors the C71G mutation associated with amyotrophic lateral sclerosis (ALS).