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.

Zeng and Madisen 2012 Prog in Brain Res

• Targeted to endogenous gene sequence disrupts or leads to fusion.

• IRES/2A add second translation site reducing protein disruption.

• Requires homologous recombination/selection.

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 recombinase

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

Cre can covalently modify DNA!

Tissue specific promoter and Cre excision

https://www.jax.org/news-and-insights/jax-blog/2011/october/cre-lox-breeding-for-dummies-part-ii

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.

Two gene systems use Cre or tTA to express

• Two gene systems DAT-Cre x Ai32 (ChR2) example (A) or Niellab GCaMP6 mouse (CaMK tTA).

• Cre is irreversible can go germline strategy B may be better, transcriptional activation versus site specific recombination.

Recombination of DNA Transcriptional activation

Two gene tTA system GCaMP6

Wekselblatt et al. 2016

CaMKII tTA

3 gene intersectional strategy specificity Ai78, Ai-93 and Ai94.

• 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

#3 Floxed and tTAdependent opsin/reporter

#1 tTA #2 Cre

GCAMP6S/F-transgenic mice Ai-93 or Ai-94 3 gene mouse!

Emx1

loxTRE lox GCAMP6S

cre

GCAMP6S

cre

STOP

Emx1-cre

Tigre-GCAMP6S

CaMKII tTA

CaMKII-tTAtTA

CaMKII-tTA Tigre-Ai-94

Emx1-cre http://connectivity.brain-map.org/transgenic/experiment/182677170

GCAMP6S

Madisen et al. 2015 Neuron

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

https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies

Jackson labs Cre cautions• 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

• 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

• 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

• 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 Creexpression 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.

• 5. There are tools to understand the level of Cre activity

• 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.

• 6. Cre can be linked to the Tet system, and vice versa

• 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.

• 7. Cre might not be active in every cell you expect

• “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.

• 8. You should not need a homozygous Cre transgene for efficient recombination

• 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.

• 9. Cre-lox can be used to create new tissue- or cell type- specific fluorescent strains

• 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.

• 10. Some floxed alleles are more easily recombined than others

• 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.

• 11. Inducible Cre can be leaky

• 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.

• 12. The Jackson Laboratory has many resources to help you with Cre-Lox

• The JAX Cre Resource is a great source of information about many strains available from The Jackson Laboratory. The Cre Portal can help you find Cre mice for your research. Not sure if your Cre is active when and where you

Zeng and Madisen 2012 Prog in Brain Res

Zeng and Madisen 2012 Prog in Brain Res