Download Transgenic approaches for optogenetics Tim Murphy UBC.

Transgenic approaches for
optogenetics Tim Murphy UBC.
• Reproducible regional expression, developmental
onset, and cell types.
• No trauma or immune response associated with viral
mediated strategies, just because only 1 protein
expressed by AAV construct does not mean microglia
will not see viral coat other proteins.
• Supports in vivo physiology and behavior.
Channelrhodopsin-2 (ChR2) transgenic mice for regional
mapping, mice express a blue light sensitive ion channel in
mostly layer 5 output neurons.
•
Transgenic mouse Thy1 ChR2 line 18 developed and tested by
Deisseroth and Feng labs (Arenkiel et al. 2007).
B6.Cg-Tg (Thy1-COP4/EYFP)
18Gfng/J mice)
Transgenic mouse issues.
• Cost of keeping line$$ and breeding downtime.
• Background strains have phenotypes for example FVB
mice are blind, C57 lose hearing as they age.
• Genotyping can be costly subject to ID errors or PCR
failure, note inquire about UBC core facilities for
genotyping Biomedical Res Centre.
• Even indicator mice GCaMP6 can have phenotypes.
• Will take months to breed Cre lines and get correct
progeny, AAV can take less than 2 weeks when injected
into an adult.
Transgenic expression strategies.
• Single promoter and reporter/opsin gene in one construct.
Advantages breeding relatively simple only works for cases with
strong promoters or where a BAC transgenic with lots of
promoter is used. Example line 18 Thy1 ChR2.
• Binary 2 gene: Cre driver two genes one inactive floxed and 2nd
gene provides Cre for site-specific recombination. DAT Cre and
Ai-32 ChR2, or 2 genes one is a transcriptional activator, tTA.
• Intersectional 3 genes: target GCaMP6 Ai94, two forms of
activator Emx-Cre, and tTA leads to increased specificity but has
overhead for breeding and genotyping. Also possible for Cre to
be expressed in development or spermatogenesis leading to
non-specific effects.
Single gene transgenic small
promoter inserts randomly in DNA.
• Construct small easier cloning example Thy-1 GFP.
• Disadvantage construct can concatenated and can
become inactivated, subject to influence by neighbor
DNA at random insertion sites.
• Requires lots of screening and luck required.
Zeng and Madisen 2012 Prog in Brain Res
Single gene promoter trap.
• Lands randomly near endogenous promoter gets
regulated by it.
• Disrupts endogenous genes difficult to predict results.
Zeng and Madisen 2012 Prog in Brain Res
Single gene BAC transgenic
• Large DNA chunk containing upstream regulatory
sequence needs to be in large content vector BAC,
example HD model mice YAC128
• Advantages very good specificity less likely to have
genome neighborhood effects, but still can be
problematic.
Zeng and Madisen 2012 Prog in Brain Res
Single gene construct targeted to
endogenous gene.
• Targeted to endogenous gene sequence disrupts or leads to fusion.
• IRES/2A add second translation site reducing protein disruption.
• Requires homologous recombination/selection.
Zeng and Madisen 2012 Prog in Brain Res
Cre recombinase
Cre recombinase is a tyrosine recombinase enzyme derived from the P1 Bacteriophage. The enzyme uses
a topoisomerase I like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of
the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between
two DNA recognition sites (LoxP sites). This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic
sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent
upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can
undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be
"floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found
between two loxP sites oriented in the same direction will be excised as a circular loop of DNA whilst intervening DNA
between two loxP sites that are opposingly orientated will be inverted.[1] The enzyme requires no additional cofactors (such
as ATP) or accessory proteins for its function.[2]
https://en.wikipedia.org/wiki/Cre_recombinase#/
Cre can covalently modify DNA!
This event can have three general outcomes based on the location and orientation of the loxP sites:
Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination
results in an inversion and the region of DNA between the loxP sites is reversed.
Deletion: If the sites face in the same direction, the sequence between the loxP sites is excised as a circular
piece of DNA (and is not maintained).
Translocation: If the sites are on separate DNA molecules, a translocation event is generated at the loxP
sites.
http://blog.addgene.org/plasmids-101-cre-lox
Tissue specific promoter and Cre excision
Examples DAT or TH Cre rats and mice used with
floxed AAV or floxed transgenic mouse
https://www.jax.org/news-and-insights/jax-blog/2011/october/cre-lox-breeding-for-dummies-part-ii
Generic Cre reporter strategy
works in first generation.
https://www.jax.org/news-and-insights/jax-blog/2011/october/cre-lox-breeding-for-dummies-part-ii
Two gene systems use Cre or tTA to express
Recombination of DNA
Transcriptional activation
• Two gene systems DAT-Cre x Ai32 (ChR2) example (A) or Niel
lab GCaMP6 mouse (CaMK tTA).
• Cre is irreversible can go germline strategy B may be better,
transcriptional activation versus site specific recombination.
Two gene tTA system GCaMP6
CaMKII
tTA
Wekselblatt et al. 2016
3 gene intersectional strategy specificity Ai78,
Ai-93 and Ai94.
#1 tTA
#2 Cre
#3 Floxed and tTA
dependent
opsin/reporter
• Dependent on the intersection of multiple factors Cre and tTA expression
• Cre can go germline complicated genotyping and breeding.
• Opsin gene or reporter usually knocked in, cloned to recombination
friendly genome area known as ROSA26 or TIGRE locus
Zeng and Madisen 2012 Prog in Brain Res
GCAMP6S/F-transgenic mice Ai-93
or Ai-94 3 gene mouse!
CaMKII-tTA
Tigre-Ai-94
Emx1-cre
http://connectivity.brain-map.org/transgenic/experiment/182677170
Madisen et al. 2015 Neuron
GCAMP6S
Emx1-cre
cre
Emx1
CaMKII-tTA
cre
tTA
CaMKII
tTA
TRE
lox
STOP
lox
Tigre-GCAMP6S
GCAMP6S
GCAMP6S
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-fordummies
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
Jackson labs Cre cautions
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1. It can matter if Cre is transmitted from the female or the male parent
•
Cre activity can vary depending if transgene is inherited from the male or female parent. For example, EIIa-Cre, B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (003724), converts floxed alleles to complete knockout alleles because Cre is
expressed throughout the mouse, but it is significantly more efficient when transmitted maternally. The Camk2a-Cre, B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (005359), recombine sequences in the hippocampus, but there is
expression in male germ cells as well. The parental inheritance pattern may not matter for all Cre strains, but it is a good idea to compare results obtained from both maternal and paternal transmission whenever possible.
•
2. Cre alone can produce a phenotype
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Cre is often expressed from a randomly integrated transgene, but very few insertion sites are known. Random transgene integration can disrupt an endogenous mouse gene and cause unanticipated side effects.
Since hemizygous mice have a wildtype chromosome present, using hemizygous instead of homozygous Cre genotypes might minimize unintended consequences of random transgene insertion. Cre toxicity can also occur when
expression is high enough to affect cell physiology, or when Cre recognizes genomic sequences that resemble loxP sites. Cre can damage DNA in fibroblasts, gastric cells, and sperm in the absence of any floxed sequence. For
these reasons, it is wise to include the Cre mouse itself (without any floxed sequences) as a control group in your experiments.
•
3. “Ubiquitous” Cre strains are not always as ubiquitous as you might think
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The tamoxifen-inducible Cre strain, B6.Cg-Tg(CAG-cre/Esr1)5Amc/J (004682), is used to conditionally delete floxed sequences throughout the entire mouse because its promoter (beta actin) should have ubiquitous
expression. Data collected by the JAX Cre Resource show that tamoxifen induction is more robust in certain tissues (pancreas and smooth muscle, for example) than others (spleen and ovary). Differences in recombination
efficiency among tissue types were also noted in a similar strain, B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J (008085).
•
4. There is a way to convert a floxed allele to a knockout, even if the mouse doesn’t carry a Cre transgene
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Oocytes in Vasa-Cre strains, FVB-Tg(Ddx4-cre)1Dcas/J (006954) and B6.FVB-Tg(Ddx4-cre)1Dcas/KnwJ (018980), express Cre mRNA/protein even when the oocyte itself does not carry the transgene. Strains with maternal Cre
expression in the oocyte can save time when converting floxed alleles to complete knockouts; no further breeding is needed to remove the Cre transgene after confirming that recombination took place. Other strains with
similar characteristics include the Sox2-cre strains: 008454, 014094, and 004783.
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5. There are tools to understand the level of Cre activity
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There is a Cre reporter, B6.129P2-Gt(ROSA)26Sortm3Nik/J (013587), that up-regulates both a red and a green fluorescent protein in cells with Cre activity. When Cre activity is especially high, however, the green fluorescence is
silenced. This tool can be used to understand the relative intensity of Cre activity in different cells or tissues.
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6. Cre can be linked to the Tet system, and vice versa
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The Tet system provides control over the timing and location of gene expression using two transgenes: a tetracycline-dependent transcription factor (tTA or rtTA) that determines tissue specificity, and a responsive promoter
(tetO) that expresses the transgene of interest. But what if you don’t have a tTA or rtTA transgenic mouse that targets your cell type, but you do have a Cre mouse with the right specificity? Mating Cre to B6.129P2(Cg)Gt(ROSA)26Sortm1(tTA)Roos/J (011008) or B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J (005670) will give you tTA or rtTA expression, respectively, in the right tissues. Similarly, there is a tetO-Cre transgene available
on C57BL/6 and BALB/c genetic backgrounds that expresses Cre if you already have a tTA or rtTA strain for your cell type.
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7. Cre might not be active in every cell you expect
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“Cre mosaicism” is documented in some strains, resulting in incomplete recombination in all cells. Data from the JAX Cre Resource have documented this in several examples.
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8. You should not need a homozygous Cre transgene for efficient recombination
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Expression levels from a hemizygous transgene should be enough to see recombination. Randomly integrated transgenes can lead to surprising phenotypes when they are bred to homozygosity.
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9. Cre-lox can be used to create new tissue- or cell type- specific fluorescent strains
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Do you need to fluorescently label a cell type, but can’t find the right transgenic mouse? If you have a Cre mouse that targets the cells you’re interested in, you can mate it with a Cre reporter to get red, green, yellow, or
even rainbow-colored fluorescence in those cells. If you prefer histology, other Cre reporters can give you beta-galactosidase (LacZ), alkaline phosphatase, or luciferase expression.
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10. Some floxed alleles are more easily recombined than others
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If the Cre-lox system is incompletely knocking out a particular gene, remember that some genes are more easily recombined than others, possibly because the chromatin state is preventing Cre from accessing the loxP sites.
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11. Inducible Cre can be leaky
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Cre is sequestered in the cytoplasm when it is fused to the ligand binding domain of the estrogen receptor, and enters the nucleus when exposed to tamoxifen. It is generally assumed that Cre is excluded from the nucleus (and
thereby unable to recombine loxP sites) in the absence of tamoxifen, but there are examples of a ubiquitous Cre, a pancreas-specific Cre and a melanocyte-specifc Cre recombining sequences without induction.
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12. The Jackson Laboratory has many resources to help you with Cre-Lox
Zeng and Madisen 2012 Prog in Brain Res
Zeng and Madisen 2012 Prog in Brain Res