Generating GFAP-Cre-ER t2 x Cx43 flox/flox Mice

3.2 Materials and Methods

3.2.1 Generating GFAP-Cre-ER t2 x Cx43 flox/flox Mice

The Gja1 gene encodes cx43, a protein abundant in astrocytes that forms the majority of their gap junctions. However, cx43 is not only expressed by astrocytes; it is essential for a synchronous heartbeat⁶³ and proper embryonic development³⁴⁰ amongst many other

functions. Cx43 knockout mice die at birth, as well. To avoid the issues inherent with a universal knockout, we generated a temporally and locally controlled knockout using the cre/lox system.

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In this system, the target gene is encapsulated within two small 34 base pair (bp) loxP sites that are carefully placed in locations that do not impede normal function; only when the protein Cre is both present and functional will the gene be excised. Thus, here we utilized a GFAP-Cre-ER^t2 mouse, which is hemizygous for a tamoxifen-controlled Cre under the GFAP promoter³⁴¹, and through a sequence of generations bred it with a mouse homozygous for flox sites surrounding exon 2 of the Gja1 gene^342,343. In the absence of Cx43, astrocytes have very few remaining gap junctions, profoundly reducing their functional connectivity. The Jackson Laboratory (Bar Harbor, ME) supplies all mice expressing a Cre-ER^T2 as hemizygotes, meaning the gene is

present on one chromosome and absent on the other (as opposed to heterozygous, where two different alleles are present). Dependent both on the location of initial gene insertion when the model is generated and the promoter used for the Cre-ER^T2, there have been reports of mice that do not survive homozygosity. However, in our model we did not see any marked

differences between mice that were hemi- or homozygous for GFAP promoted Cre-ER^T2,

including rates of gene excision. For this reason, we kept all mice that had any expression of cre in experimental groups, and later genotyped for gene excision in every mouse in target tissue to confirm the knockout.

The initial cross utilized six mice, one male and two females each of mice hemizygous for GFAP-Cre-ER^t2 or homozygous for cx43^flox/flox. A male of one genotype was caged in a harem with two females of the opposing genotype to maximize efficiency and minimize cost at this early stage. Crosses resulted in a 1 in 2 chance of the desired genotype, which at this stage is a mouse hemizygous for a GFAP-Cre-ER^t2 and heterozygous for Cx43^flox (Figure 3.1).

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For the second cross, we utilized two strategies to maximize the number of usable mice in our colony and increase output of potentially useful offspring (Figure 3.2). For both of these crosses, we are targeting

mice positive for cre (whether hemi- or homozygous) and

homozygous for our floxed gene. We only need cre to be present to achieve our knockout; however, the Gja1 genes on both alleles must be floxed to eliminate the presence of Cx43 protein.

The first strategy involved crossing two unrelated offspring from cross 1, both

of which are positive for cre and heterozygous for floxed Cx43. This cross resulted in three of 16 offspring with our target genotype. The second strategy utilized parent 2 from cross 1, a mouse without cre but homozygous for floxed Cx43, and an unrelated mouse positive for cre and heterozygous for floxed Cx43. This cross resulted in one of four offspring with our target genotype, and increased the number of breeder cages to provide more potential unrelated mice for future crosses.

Cre⁺ flox^- Cre⁺ flox^- Cre^- flox^- Cre^- flox^-

Cre^- flox⁺ Cre^+/-, flox^+/-Cre^+/-, flox^+/- Cre^-/-, flox^+/- Cre^-/-, flox^+/-

Cre^- flox⁺ Cre^+/-, flox^+/-Cre^+/-, flox^+/- Cre^-/-, flox^+/- Cre^-/-, flox^+/-

Cre^- flox⁺ Cre^+/-, flox^+/-Cre^+/-, flox^+/- Cre^-/-, flox^+/- Cre^-/-, flox^+/-

Cre^- flox⁺ Cre^+/-, flox^+/-Cre^+/-, flox^+/- Cre^-/-, flox^+/- Cre^-/-, flox^+/- Parent 2:

Cre^-/-, flox^+/+

Parent 1:

Cre^+/-, flox^-/- Cross 1

Figure 3.1 Generating GFAP-Cre-ER^t2 x Cx43^flox/flox mice, cross 1.

In our initial cross, we utilized a mouse hemizygous for GFAP-Cre-ER^t2 (parent 1) and crossed with a mouse homozygous for Cx43^flox. The half of offspring positive for cre and heterozygous for floxed Cx43 were utilized in cross 2 (bold, white offspring).

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Once we obtained an unrelated male and female with our target genotype from cross 2 we began cross 3, the final cross in our sequence and the cross resulting in our experimental mice. Because our target genotype from cross 2 involves mice positive for GFAP-Cre-ER^t2, whether homo- or hemizygous, and our genotyping method (detailed in section 3.2.2) only reveals the presence of GFAP-Cre-ER^t2 and not its zygosity, crosses here had variable results dependent on the true genotype of both parents. In all cases offspring were now homozygous for Cx43^flox, as we were breeding two mice homozygous for this gene. If both parents were hemizygous for GFAP-Cre-ER^t2, half of offspring were positive for GFAP-Cre-ER^t2 while the other half were cre negative; these cre negative mice were then utilized as littermate controls for

Cre⁺ flox⁺ Cre⁺ flox^- Cre^- flox⁺ Cre^- flox^- Cre⁺ flox⁺ Cre⁺ flox^- Cre^- flox⁺ Cre^- flox^-

Cre⁺ flox⁺ Cre^+/+, flox^+/+Cre^+/+, flox^+/-Cre^+/-, flox^+/+Cre^+/-, flox^+/- Cre^- flox⁺ Cre^+/-, flox^+/+Cre^+/-, flox^+/-Cre^-/-, flox^+/+ Cre^-/-, flox^+/-

Cre⁺ flox^- Cre^+/+, flox^+/-Cre^+/+, flox^-/- Cre^+/-, flox^+/- Cre^+/-, flox^-/- Cre^- flox⁺ Cre^+/-, flox^+/+Cre^+/-, flox^+/-Cre^-/-, flox^+/+ Cre^-/-, flox^+/-

Cre^- flox⁺ Cre^+/-, flox^+/+Cre^+/-, flox^+/- Cre^-/-, flox^+/+ Cre^-/-, flox^+/- Cre^- flox⁺ Cre^+/-, flox^+/+Cre^+/-, flox^+/-Cre^-/-, flox^+/+ Cre^-/-, flox^+/-

Cre^- flox^- Cre^+/-, flox^+/- Cre^+/-, flox^-/- Cre^-/-, flox^+/- Cre^-/-, flox^-/- Cre^- flox⁺ Cre^+/-, flox^+/+Cre^+/-, flox^+/-Cre^-/-, flox^+/+ Cre^-/-, flox^+/- Cross 2a

Parent 1:

Cre^+/-, flox^+/-

Parent 2:

Cre^+/-, flox^+/-

Cross 2b

Parent 1:

Cre^+/-, flox^+/-

Parent 2:

Cre^-/-, flox^+/+

Figure 3.2 Generating GFAP-Cre-ER^t2 x Cx43^flox/flox mice, crosses 2a and 2b.

In our second cross, we utilized two strategies to optimize the number of available target offspring, mice positive for cre (whether hemi- or homozygous) and homozygous for Cx43^flox (bold, white offspring). In cross 2a (left), both mice are unrelated offspring from cross 1, positive for cre and heterozygous for floxed Cx43. This cross resulted in 3 of 16 mice with our target genotype. Cross 2b involved one offspring from cross 1 (Parent 1) and an unrelated mouse without cre but homozygous for Cx43^flox (Parent 2), resulting in 1 of 4 offspring with the target genotype.

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tamoxifen induction and genetic manipulation. If either parent was homozygous for GFAP-Cre- ER^t2 all offspring were cre positive and were viable for experimental cohorts.

All mice detailed herein were generated on a C57BL/6 (C57) background, so C57 mice (Jackson Labs, Bar Harbor, ME) were always used as a wild-type control.