Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR)

Abstract

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Figure 1

Schematic of small DNA fragment (SDF) generation and PCR analysis of SFHR A. SDF (783bp) containing the ΔF508 mutation and a KpnI restriction enzyme cleavage site was synthesized using primers mCF1 and mCF15, localized within introns 9 and 10 of Cftr gene respectively (2). The unique KpnI site is a secondary marker for assessment of SFHR-mediated modification. B. Analysis of genomic DNA using two successive rounds of PCR amplification. The first round of PCR used primers (mCFf/mCFr) were located outside the region of homology defined by the SDF and resulted in a 1080- or 1077-bp amplicon (wild tyoe of ΔF508, respectively). The second round of amplification used the amplicon generated in the first round as a template and allele specific primers (mCF4/mCF3N or mCF4/mCFΔF for wild type and ΔF508 sequence respectively). The 478-bp fragment was then digested with KpnI to determine whether the 442- and 36-bp restriction fragments, indicating SFHR mediated replacement, were present. C. Allele specific RT-PCR for analysis of transfected ES cells using primers located within exon 9 (mCF11) and exon 10 (mCF ΔF or mCF3N, wild type and ΔF508 respectively). The 237- or 234-bp amplicon was then digested with KpnI to assay for restriction fragments of 198-bp and 36-bp indicating expression of SDF-derived sequences.

Figure 2

Polyacrylamide gel analysis of allele-specific PCR amplification products generated from the genomic DNA of transfected cells and digested with KpnI. Lane M: 50-bp DNA ladder (Invitrogen, Carlsbad, CA). Lanes 1-6: amplicons derived from ES cells transfected with different quantities of SDF; lane 1 and 2 correspond to cells transfected with 6.4 × 10⁵ SDF/cell, lane 3 and 4 with 1.2 × 10⁶ SDF/cell, and lane 5 and 6 with 1.9 × 10⁶ SDF/cell. Lanes 1, 3 and 5 are amplicons obtained with primers mCF4/mCF3N while lanes 2, 4 and 6 with primers mCF4/mCF3ΔF. Ctr sample is derived from ES cells transfected with Smn-SDF (8). All samples amplified with primers mCF4/mCF3N and mCF4/mCF3ΔF and digested by KpnI. The 442-bp band is the result of KpnI digestion of SFHR-modified genomic DNA. Arrows indicate the molecular weight of amplicons and of its digestion products.

Figure 3

Gel electrophoresis analysis of AS-PCR performed on mRNA-derived cDNA from transfected ES cells. Wild type and ΔF508 amplicons were generated using primers mCF11/mCF3N and mCF11/mCF3ΔF, respectively (see Figure 1 C). These amplicons were cloned into vectors which were then isolated clones and sequenced. Sequencing of mCF11/mCF3ΔF (MUT) exclusively showed the presence of both the ΔF508 allele and the KpnI restriction site (arrows) indicating that the SDF-derived sequences were expressed as CFTR mRNA.

Figure 4

Quantitative PCR analysis of the ΔF508 and wild type Cftr transcript in transfected D3 ES cells. Open columns represent the wild type transcript, while shaded columns represent the ΔF508 transcript. Sample 1 corresponds to cells transfected with no SDF, sample 2 to cells transfected with SDF homologous to Smn gene, and samples 3 to cells transfected with 1 × 10⁶ ΔF508-SDF/cell. The values obtained from treated cells represent the mean of at least three independent experiments that were performed in triplicate. Values were significantly different from those obtained with untreated cells. Error bars indicate the SD. A p value of < 0.05 was considered statistically significant.

Figure 5

CFTR-dependent chloride efflux of ES-D3 (A) and electroporated ES-D3 (B) cell populations. Typical recordings (left panels) obtained by spectrofluorimetric analysis of the entire ES-D3 and electroporated ES-D3 populations seeded on glass coverslips (see Methods) show the changes in intracellular Cl--dependent MQAE fluorescence (expressed as the F/F0 ratio) when the cells were treated with 10 μM FSK plus 500 μM IBMX following substitution of chloride by nitrate in the absence or presence of 100 μM glibenclamide. Glibenclamide was applied before the next FSK+IBMX stimulation and remained during the entire chloride efflux measurement. The right panels show the summary of data from n=14 and n=13 experiments for ES-D3 (A) and electroporatedd (B) ES-D3 cells respectively. The glibenclamide-sensitive, CFTR-mediated chloride efflux rates (empty bars) were calculated as the difference in the F/F0 ratio per minute ((F/F0)/min) in the absence of (light gray bar) and presence of (dark bar) glibenclamide. Each bar represents the mean ± S.E. The data were compared by using the two-tailed, paired Student's t test analysis. p < 0.05 was considered statistically significant.