Online Methods for MS#829-R2:
Cloning of Kir 2.1 and Kir 2.2 and generation of targeting constructs
Mapped 129S/v murine genomic plasmids (courtesy of Dr. Bruce Tempel University of Washington, Seattle) included the entire coding sequence for the Kir2.1 protein and significant regions of flanking non-coding sequence. The murine Kir2.2 gene was isolated from a 129S/v mouse genomic library with an 892 bp probe from the Kir2.2 open reading frame (bp 250-1141).
The 5’ arm of the Kir2.1 targeting construct was a ~5.2 kb Sac I fragment of the genomic clone just upstream of the Kir2.1 open reading frame. A 3.5 kb Pst I fragment downstream of the cDNA coding sequence was used for the 3’ arm (Figure 1a). For Kir2.2, a 2.5 kb Not I-BamHI fragment 900 bases upstream from the beginning of the Kir2.2 open reading frame made up the 5’ arm while the 3’ arm consisted of a 7.2 kb Stu I- Sma I downstream fragment (Figure 1b). In both constructs, the neomycin resistance gene, driven by the phosphoglycerate kinase promoter, was interposed between the two arms. A thymidine kinase cassette, also driven by that promoter, was placed after the 3’ arm.
ES cell electroporation
Linearized gene targeting constructs were introduced into R1 ES cells (courtesy of Andras Nagy, Mount Sinai Hospital, Toronto, Canada) using standard electoporation techniques 1. After a single discharge of 500 µF, 250 V (Gene Pulser electroporator, Bio-Rad) cells were allowed to recover at room temperature for 10 minutes before being plated onto 4 six well tissue culture plates with confluent murine embryonic fibroblast feeder cells. After 24 hours, transfected cells were selected with 200 µg/ml G418 (Geneticin, Gibco) and 2 µmol/L gancyclovir (Cytovene, Roche). Media was changed daily and selection continued for 7 days. Colonies were picked manually, trypsinized, and then plated onto 96 well tissue culture plates. The colonies were allowed to expand and then were split onto (2) 96 well plates. One plate was frozen at –80° C and the other replica plate was used to isolate genomic DNA from each clone. Homologous recombination events were determined by the hybridization pattern on a genomic Southern blot. Blots were loaded with 15 µg of genomic DNA per clone and transferred onto nylon. DNA from Kir2.1 targeted cells was digested with Afl II and probed with a 2.2 kb fragment 3’ to the targeting vector (Figure 1a). DNA from Kir2.2 targeted cells was digested with BamHI and probed with a 0.9 kb fragment 3’ to the Kir2.2 targeting vector (Figure 1b).
Generation of Targeted Mice
ES cells heterozygous for the Kir2.1 targeting vector were injected into blastocysts of C57BL/6 mice (University of Cincinnati) and highly chimeric mice were mated to NIH Swiss Black females. Germ line transmission to offspring was confirmed by Southern analysis of tail DNA. Chimeric Kir2.2 mice were generated by aggregation with morulae from CD-1 mice 2. Chimeras were mated to non-agouti CD-1 females and germline transmission was determined as above.
Tissue Preparation
Kir2.1-/- or control littermates (Kir2.1+/+ and Kir2.1+/-) mice (< 1 day postnatal) of either sex were euthanized by exsanguination while under deep pentobarbital anesthesia (intraperitoneal; 150 mg kg-1 body weight). Because the cleft palate was shown to be 100% indicative of the Kir2.1-/- genotype, homozygous animals were selected for recordings by examination of the palate. Cerebral arteries were rapidly dissected while the brain was submersed in cold (4°C) oxygenated (95% O2/ 5 % CO2) physiological saline solution (PSS) of the following composition (in mmol/L): 118.5 NaCl, 4.7 KCl, 24 NaHCO3, 1.18 KH2PO4, 2.5 CaCl2, 1.2 MgCl2, 0.023 EDTA, 11 glucose. Cerebral arteries from adult Kir2.2-/- animals (16 to 24 weeks) or similarly aged controls (either FVB or Kir2.2+/+ littermates) were obtained in a manner similar to that described above for neonatal mice.
Diameter Measurements in Isolated Arteries
Cerebral artery segments were cannulated on glass pipettes mounted in a 5 ml myograph chamber. The intravascular pressure of arterial segments was increased from 3 mm Hg up to 40 mm Hg at the start of each experiment. Pressurized arteries were continuously superfused with PSS aerated (95% O2/ 5% CO2) at 37° C and pH 7.4 3. Arterial diameter was measured with video edge detection equipment and recorded using data acquisition software developed by IonOptix Inc. (Milton, MA, USA). Analysis was made using Ion Wizard (IonOptix Inc., Miltion, MA, USA).
Pressure-induced constrictions are expressed as a percent decrease of the fully dilated diameter of individual arteries at the same intravascular pressure. These values were obtained by using the following equation:
where DP = the (passive) diameter of the artery in Ca2+-free PSS containing 1 µmol/L nisoldipine, and DA = the (active) diameter of the artery in response to the stimulus in Ca2+-containing PSS.
Dilations in response to elevated extracellular K+ (15 mmol/L) or forskolin (1 µmol/L) are expressed as a percent reduction in the level of arterial constriction. The values were obtained from the following equation:
where DV = arterial diameter in the presence of vasodilator, DA = the active diameter of the artery prior to addition of vasodilator, and DP = the passive diameter of the artery.
K+ Current Recordings
Smooth muscle cells from cerebral arteries were enzymatically isolated in a manner similar to that described previously 4. Whole-cell K+ currents in freshly isolated cerebral artery myocytes from neonatal mice were measured using the conventional whole cell configuration of the patch clamp technique 5. Patch pipettes (resistances, 3-5 MW) were filled with a solution containing (in mmol/L): 87 KAsp, 20 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 EGTA, 25 KOH (pH 7.2). Following the establishment of whole-cell configuration, the mean series resistance was 6.1 ± 0.6 MW (n=25) and 8.1 ± 0.8 MW (n=23) in cells from control and Kir2.1-/- animals, respectively. Seals were made in an extracellular solution containing (in mmol/L): 134 NaCl, 6 KCl, 1 MgCl2, 0.1 CaCl2, 10 glucose and 10 Hepes (pH 7.4). To maximize the inward rectifier K+ currents, extracellular K+ was raised to 140 mmol/L (inside, 140 mmol/L K+). To compensate for differences in cell size, membrane K+ currents are expressed relative to cell capacitance (pA/pF). To exclude membrane K+ currents resulting from the activation of KATP channels, glibenclamide (10 µmol/L) was included in the extracellular solution.
Data Analysis
Data are expressed as mean ± SEM. Statistical significance (p<0.05) was assessed using Student’s paired or unpaired t-test as appropriate.
References for Online Methods:
1. Joyner AL. Gene targeting : a practical approach. Oxford ; New York: IRL Press at Oxford University Press; 1993.
2. Wood SA, Allen ND, Rossant J, Auerbach A, Nagy A. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature. 1993;365:87-9.
3. Knot HJ, Zimmermann PA, Nelson MT. Extracellular K(+)-induced hyperpolarizations and dilatations of rat coronary and cerebral arteries involve inward rectifier K(+) channels. J Physiol (Lond). 1996;492:419-30.
4. Perez GJ, Bonev AD, Patlak JB, Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol. 1999;113:229-38.
5. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.