The kick-in system: a novel rapid knock-in strategy

Abstract

Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.

Figure 1. Comparison of Kick-in and Knock-in methodologies.

On the left is a schematic of a traditional knock-in along with the estimated duration of each step. Juxtaposed on the right are the corresponding steps for a kick-in; savings in time and cost versus the knock-in are highlighted by a little stopwatch. Low recombination rates (∼1∶10⁵) are associated with homologous recombination. To make several constructs, homologous recombination must be performed for each construct. In the kick-in system, homologous recombination is needed only once, even if several constructs are made.

Figure 2. The Kcnq2 kick-in.

(A) Depicted is the murine Kcnq2 gene. Introns are shown as a solid horizontal line and the interspaced white bars symbolize the numbered exons. Aligned underneath is the targeting vector construct. Areas of homologous recombination are indicated by crosses between the two DNAs. The targeting vector, which confers neomycin resistance, introduces various lox elements and flippase recognition targets (FRTs) that are primed for subsequent recombination exchange with a puromycin-resistance mutation vector that contains the desired missense variations in exon 6. Triangular symbols are explained in Table 1. Additional detail is provided in the Methods & Materials. (B) Schematic explaining PCR and Southern blot analyses confirming homologous recombination in cloned embryonic stem cells. Arrows at the bottom demark the positions of primers used in the PCR analysis. Double-headed arrows span the restriction fragment used in the Southern blot analysis with the probe shown at the bottom. (C) Identification of homologous recombination clones. Top: PCR amplicons generated using the PCR primers that span the wildtype/recombination junction (see Panel B), such that only mutant DNA is expected to be amplified; agarose/TBE gel with the expected band at 1.8 kb highlighted with an asterisk (*); other bands result from non-specific amplification. Bottom: To confirm that the PCR-amplified 1.8-kb fragment originated from the recombinant Kcnq2 allele, the DNA was analyzed by Southern blot hybridization with the probe shown in Panel B. (D) Southern blot of the genomic DNA restriction fragments from six targeted clones. Presence of a 4.4-kb signal indicates wildtype DNA, whereas 3.2-kb confirms mutant DNA (irrespective of Y284C or A306T mutation). Control setups used wildtype DNA from C57BL/6J wildtype mice.

Figure 3. Genotyping of Kcnq2 mutant mice.

(A) Based on structural analogies to related channels, p.Tyr284 and p.Ala306 map to the outer mouth of the pore and the inner lining of the channel, respectively. (B) Expression specificity of the mutated Kcnq2 alleles. Exon 6, including 350-bp segments of Kcnq2 transcripts from various tissues, was amplified by RT-PCR and electrophoresed on 2.0% agarose/TBE gels (top). Reference RNA samples are shown at the bottom. Br, brain; Ht, heart; Lg, lung; Lv, liver; Sp, spleen; Kd, kidney; Ts, testes; Sm, skeletal muscle. (C) Nucleotide sequence of wildtype and knock-in DNA. Direct sequencing of a PCR fragment amplified from genomic DNA using primers complementary to the lox71/KMR3 site and the FRT site, which are 5′ and 3′ of exon 6, respectively. Depicted are partial chromatograms for nine bases that cover the codon of interest, as well as one upstream and one downstream codon at position p.Tyr284 (top) and p.Ala306 (bottom). From left to right are sequencing results for wildtype, and heterozygotes of Y284C and A306T. Arrowhead, mutated base.

Figure 4. Morphological studies.

(A) Light micrographs of K_v7.2 immunoreactivity in the sensorimotor cortex. Note the immunostained neuronal cell bodies in layers II to VI. (B) Cell count of K_v7.2-immunopositive neurons in layers II/III and V in the frontal cortices of wildtype, heterozygous, and homozygous Y284C mice. No differences were noted.

Figure 5. Behavioral analysis of Kcnq2 kick-ins.

(A) Electroencephalography (EEG) with video monitoring revealed spontaneous myoclonic seizures with spike discharges in all Y284C homozygotes (n = 4). On average, the animals had 12 myoclonic seizures per hour in the daytime. (B) Modified Racine’s scale. Behavioral criteria used to score the seizures in this study. Adopted and modified from Lüttjohann et al., 2009. All seizures scoring 4 or above were generalized. (C) Pentylenetetrazole/saline (PTZ) induced seizures in wildtype (white bar), Y284C, and A306T heterozygous mice (grey bars). The bar height represents the average seizure score; the hashed part of the bar represents the fraction of the animals that reached seizure score 4 and above. Asterisks denote P<0.05 (Tukey-Kramer test).

Figure 6. Kcnq2 mutant brain slice electrophysiology.

(A) Sample traces recorded from 10-week old Y284C heterozygous CA1 neurons in response to voltage steps to the indicated potential. (B, C) Voltage-dependence of M-current activation in wildtype (○), heterozygous (), and homozygous (•) Y284C mice at 4 weeks (B) and 10 weeks (C). Whole-cell perforated patched CA1 neurons were stepped to the indicated potential and back to −20 mV to determine tail current amplitudes. Significant I _M reductions were seen in Y284C homozygotes at 4 and 10 weeks, and in heterozygotes at 10 weeks at −60 mV (P<0.05, Tukey-Kramer test).