Molecular Vision 2000; 6:85-94 <>
Received 26 April 2000 | Accepted 30 May 2000 | Published 2 June 2000

Lim2To3 transgenic mice establish a causative relationship between the mutation identified in the Lim2 gene and cataractogenesis in the To3 mouse mutant

Ernest C. Steele, Jr.,1 Jian-Hua Wang,1 Woo-Kuen Lo,2 David A. Saperstein,1 XiaLian Li,1 Robert L. Church1

1Emory Eye Center, Emory University School of Medicine, Department of Ophthalmology, Atlanta, GA; 2Department of Anatomy, Morehouse School of Medicine, Atlanta, GA

Correspondence to: Robert L. Church, Ph.D., Emory Eye Center, Room B5601, 1365B Clifton Road, NE, Atlanta, GA, 30322; Phone: (404) 778-4101; FAX: (404) 778-2232; email:


Purpose: Lim2 is the gene encoding the ocular lens-specific intrinsic membrane protein MP19. We previously reported finding a single nonconservative G->T transversion in exon two of the Lim2 gene. This mutation was linked to the cataract in the To3 (Total opacity number 3) mouse mutant, confirming Lim2 as an ideal candidate gene for the To3 cataract. The aim of the present study was to substantiate a causative relationship between the mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. To this end a Lim2To3 transgene cassette was engineered and introduced into fertilized normal mouse embryos to test its ability to induce cataractogenic lens development.

Methods: A Lim2 genomic clone was isolated and purified from a murine 129/SvJ genomic library. A restriction endonuclease map of the gene was generated using classical Southern techniques. The murine Lim2 promoter was characterized by transfecting primary chicken lens epithelial cells with Lim2 promoter-CAT reporter constructs and assaying promoter activity and specificity. This genomic clone was then used in conjunction with PCR to generate a Lim2To3 transgene cassette. After sequencing of the PCR engineered portion, the Lim2To3 transgene was then used to generate Lim2To3 transgenic mice via pronuclear injection. Founder mice and their offspring from outcrosses and intercrosses were characterized by ophthalmic examination, PCR and Southern DNA analysis, RT-PCR mRNA analysis, and histology of lens sections.

Results: Two mice, from independent microinjections, were identified as positive for presence of the Lim2To3 transgene cassette as well as presence of bilateral congenital cataracts and reduced eye size and mass. One of these founders was incapable of germline transmission of the transgene to offspring and was not characterized further. The other was capable of germline transmission and was characterized as described above. PCR DNA analysis revealed a perfect concordance between presence of the Lim2To3 transgene cassette and congenital cataract in offspring of this founder. Transgenic hemizygotes exhibited cataract and a reduction in eye and lens size and mass, while transgenic "homozygotes" presented with a more severe cataract and microphthalmic reduction in eye and lens size and mass. Southern analysis revealed approximately 2 copies of the transgene cassette integrated into a single chromosomal site in the founder and all hemizygous offspring. RT-PCR analysis revealed a very low ratio of Lim2To3 transgenic mRNA compared to endogenous normal Lim2. Finally, histology revealed that lens development was abnormal in mutant transgenic animals by embryonic day E15. By E19, just prior to birth, gross disorganization of secondary fibers was observed in mutants.

Conclusions: These transgenic experiments firmly establish a causative relationship between the previously identified mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. The low levels of mutant mRNA produced by the transgene cassette as compared to endogenous levels of normal Lim2 mRNA provides evidence that this dominant mutation results in a mutant MP19 protein with altered function rather than simply loss of function.


In the mammalian lens fiber cell, the vast majority of the intrinsic membrane proteins present are represented by four different proteins, termed (for historical reasons) MP19, MP26, MP46, and MP70. MP46 and MP70 are in the connexin family and MP26 (or MIP) is probably a member of the water channel class of membrane proteins. MP19 is the second most abundant integral membrane protein in lens fibers. It was first described as a fiber-specific component of bovine lens membranes [1] and a cDNA encoding this protein was subsequently cloned from a bovine lens cDNA library [2]. The entire human gene, termed LIM2 [3] has since been cloned and reported.

MP19 is phosphorylated by cAMP-dependent protein kinase and appears to bind calmodulin [1,4,5]. These features are indicative of a protein with a function that can be modulated. However, MP19 bears no striking resemblance to any other reported proteins and has no clearly defined structural or functional role. MP19 is uniformly distributed throughout fiber membranes, and also colocalizes with gap junctions in distinct regions of the lens [6,7]. It is therefore possible that MP19 may play some role in gap junction formation, maintenance or organization.

We previously reported the identification of a single G->T transversion within exon 2 of the Lim2 gene in the To3 mouse mutant [8], which was linked to cataractogenesis and severe microphthalmia. This mutation is predicted to result in a nonconservative amino acid substitution within the MP19 polypeptide, the protein encoded by the Lim2 gene. In that communication, we touted this mutation as the underlying molecular genetic defect in the To3 mouse. However, we also cautioned that the data was only intriguingly suggestive of a causative relationship and far from proof. For further substantiation, we proposed to engineer a Lim2To3 transgene carrying the To3 mutation and to test its ability to induce cataractogenesis and microphthalmia in transgenic mice. We have since executed these experiments, as are described below. These data strongly support our previous extrapolations of a causative relationship between the mutation and cataractogenesis. Furthermore, they establish Lim2 as a promising candidate gene for human hereditary cataract with a known but unidentified genetic basis.


Chemicals and Enzymes

Unless otherwise noted, all chemicals used to prepare buffers and solutions were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Unless otherwise noted, all enzymes employed in DNA cloning and restriction analysis were purchased from Life Technologies (Gaithersburg, MD).

Isolation of a murine Lim2 genomic clone

A 129/SvJ mouse genomic DNA library constructed using liver genomic DNA and the Lambda Fix II vector was obtained from a commercial vendor (Stratagene, La Jolla, CA). This library was titered and ~1 x 106 clones were plated using 150 mm petri dishes at ~50,000 pfu per plate according to the manufacturer's protocols. Replica filters were prepared using 137 mm Magna-Graph nylon transfer membranes (Micron Separation, Inc., Westborough, MA).

The probe used for screening the library was a 500 bp cDNA encoding the entire bovine MP19 polypeptide [2]. A radiolabeled probe was generated from this cDNA using the Random Primers Radiolabeling kit (Life Technologies) and [α-32P]-dCTP (Amersham Corp., Arlington Heights, IL) according to the manufacturer's protocol.

Filters were prehybridized in an excess of buffer (50% deionized formamide, 0.2% SDS, 4X SSC, 0.1% sodium pyrophosphate, and 50 μg/ml heparin) for approximately 4 h at 42 °C in a Gene Roller hybridization oven apparatus (Savant, Farmingdale, NY). Filters were then hybridized overnight at 42 °C in the above hybridization buffer containing probe at a final concentration of approximately 1 x 106 incorporated CPM/ml. The filters were washed three times for one h each in excess 0.1% SDS/0.1X SSC buffer at 65 °C. The filters were then exposed using the GS-525 Molecular Imager System (Bio-Rad Laboratories, Hercules, CA).

A single positive plaque was identified, picked and purified through three successive platings and screenings. Purified phage were used to prepare lysates from which phage DNA was isolated using the Wizard Lambda Preps DNA Purification Kit (Promega, Madison, WI) according to manufacturer's protocols.

Generation of murine Lim2 restriction endonuclease map

Phage DNA purified as described above was initially characterized by digestion using the following restriction endonucleases: BstE II, BamH I, Xho I, Xba I, Pst I, Hind III, Kpn I, Dra I, Stu I, Sst I, Sst II, EcoR I, EcoR II, Eco RV, Sma I. Restricted DNAs were electrophoresed on a 0.7% agarose gel and Southern blot analysis of the restriction fragments using 5' (exon 2)- and 3' (exon 5)- specific probes. This allowed the development of a restriction map of the entire murine Lim2 gene. The original clone contained an ~18 kb insert. The insert was determined to contain ~400 bp of 5' upstream sequence, the entire MP19 protein coding region and ~11 kb of 3' downstream sequence.

Trimming and Subcloning of murine Lim2 genomic clone

The restriction map of the original insert indicated a single BamH I restriction site ~11 kb from the 5' end (~400 bp upstream of the Lim2 transcription start site) and ~5 kb past the 3' (poly A site) end of the gene. This ~11 kb fragment was released using Not I (within the 5' end of the lambda vector multiple cloning site) and BamH I. This DNA fragment was purified by gel electrophoresis and recovered from agarose using the Wizard DNA Cleanup System (Promega). The purified DNA fragment was subsequently subcloned between the corresponding sites within the multiple cloning site of the pBluescript II SK- bacterial plasmid vector (Stratagene) using standard subcloning techniques. Subsequent sequencing across the 3' end (using methods described below) of the Lim2 gene revealed a single Avr II restriction site just past the consensus polyadenylation signal of the Lim2 gene. Sst I (within the 5' multiple cloning site of the lambda vector) was used along with Avr II to generate an ~6.3 kb restriction fragment encompassing the entire Lim2 gene. This fragment was purified as before and subsequently subcloned between Sst I and Xho I (compatible with the ends generated with Avr II) of the pBluescript II SK-plasmid vector using standard subcloning techniques.

DNA Sequencing

Primers were designed based upon the human Lim2 sequence [3] and were used to sequence the entire Lim2 promoter region, all exons and flanking splice sites, and the 3' end of the gene. Plasmid DNA was used as template in cycle sequencing reactions using Cy5-labeled oligonucleotide primers and the Cy5 AutoCycle Sequencing Kit (Pharmacia Biotech, Piscataway, NJ). These reactions were then electrophoresed and analyzed using an ALFexpress automated DNA sequencer (Pharmacia Biotech). Nucleotide sequence and predicted amino acid sequence analysis was performed using DNASIS and PROSIS software (Hitachi America, Ltd., Brisbane, CA).

Characterization of murine Lim2 promoter

Various fragments of the murine Lim2 promoter region, all terminating at the end of exon 1 at the 3' end and having different starting points within the upstream sequence, were amplified from the pBluescript II SK-/ To3 transgene construct via PCR using the proofreading Pfu DNA polymerase (Stratagene). These were then subcloned into the promoterless CAT reporter vector pSVOATCAT [9].

CAT assays were carried out using the general procedures of Chepelinsky [10] and Klement et al. [11] with the modification of Cassinotti and Weitz [12], to improve sensitivity. Transient transfections were carried out as described by Klement et al. [11], using lipofectAMINE (Life Technologies) instead of calcium phosphate. The chick embryo lens epithelial cell cultures were used as described by Borras et al. [13]. Cells were co-transfected with the plasmid pIC 409 containing the β-Gal gene to monitor transfection efficiency. The cells were harvested 72 h after transfection and assays for CAT and β-Gal activity were carried out.

Engineering of Lim2To3 transgene cassette

PCR was used to amplify the 5' fragment of the transgene cassette from To3/To3 genomic DNA. Primers used contained restriction sites (Sst I on the 5' sense primer and Dra I on the 3' antisense primer) to facilitate the cloning process. The ~2.3 kb amplification product spanned from the -399 nucleotide position in the promoter through exons 1 and 2, and terminated at the Dra I site within intron B. The fragment was then digested with Sst I to generate a Sst I cohesive end. This final fragment was then ligated with an ~4 kb Dra I -> BamH I restriction fragment generated from the 6.3 kb insert described above and pBluescript II SK- (digested with Sst I and BamH I). The PCR engineered portion of the final pBluescript II SK-/To3 transgene clone was then sequenced as described below to verify the presence of the To3 mutation within exon 2 and the lack of other sequence alterations within the exons and splice junctions.

Generation of Lim2To3 transgenic mice

Digestion of the pBluescript II SK-/To3 transgene construct with Sst I and BamH I generated the To3 transgene DNA. The liberated ~6.3 kb DNA cassette was then separated on a preparative agarose gel with TAE buffer. The fragment was then electroeluted using an Elutrap device (Schleicher & Schuell, Inc., Keene, NH). Following volume reduction by n-butanol extraction, the DNA was precipitated overnight at -20 °C with 0.1 volumes of 3 M sodium acetate and 2 volumes of 100% ethanol. The DNA pellet was then washed 2 times with 70% ethanol at room temperature for 10 min and then air-dried at room temperature. DNA was resuspended in injection buffer (10 mM Tris [pH 7.4], 0.1 mM EDTA) and then passed through a Millipore Ultrafree-MC ProBind spin filter (Millipore Corporation, Bedford, MA) to remove any particulate matter and protein.

This DNA was then used by the Emory University Winship Cancer Center Transgenic Facility to produce potential transgenic founder mice via microinjection of fertilized single-cell (C57BL/6X SJL)F2 hybrid mouse eggs. Surviving eggs were then implanted into pseudopregnant recipient females and delivered to term.


All animals were treated in compliance with the Guiding Principles in Care and Use of Laboratory Animals (DWEH Publication, NIH 86-23) and according to the tenets of the ARVO Statement for the use of Animals in Ophthalmic and Vision Research.

PCR screening of potential transgenic mice

Tail clippings (1-1.5 cm) were taken from weanling mice (14-20 days old) and genomic DNA was isolated using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). A ~2.2 kb fragment encompassing exon two and flanking sequences was amplified using the proofreading Pfu DNA polymerase (Stratagene) and Lim2-specific primers as described previously [8]. Subsequent digestion of the amplification product with BstE II (the restriction site created by the mutation identified in the Lim2 gene of the To3 mouse) resulted in digestion of transgenic amplicon DNA, allowing it to be distinguished from endogenous Lim2 amplification product.

Ophthalmoscopic screening of the transgenic mice

Mice were scored for the presence of cataracts using slit lamp biomicroscopy and indirect ophthalmoscopy with a 78 diopter aspheric lens (Volk, Mentor, OH) following dilation of the iris with 0.5% tropicamide (Mydriacyl from Alcon Inc., Humacao, Puerto Rico, USA) and 2.5% phenylephrine hydrochloride (AK-Dilate from Akorn, Inc., Abita Springs, LA) solutions.

Light microscopy

Embryonic mouse eyes and freshly isolated lenses of postnatal mice were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) containing 50 mM L-lysine and 1% tannic acid [14] for 2-4 h or overnight. Fixed eyes and lenses were postfixed in 1% aqueous OsO4 for 1 h, dehydrated through graded ethanol and propylene oxide, and embedded in Polybed 812 resin (Polysciences, Warrington, PA). Thick sections (1-1.5 μm) were stained with 1% toluidine blue and examined and photographed with a Zeiss Axiosckop using Kodak TX400 film.

Southern and dot blot DNA analyses

The probe (a 500 bp cDNA encoding the entire bovine MP19 polypeptide) and hybridization techniques employed in all Southern procedures was as described above for the isolation of the murine genomic Lim2 clone. Following digestion with BamH I and Stu I, genomic DNA was electrophoresed on a 0.7% agarose gel and analyzed using Southern blot analysis to determine the number of transgenic DNA integration sites. Following digestion with EcoR I, genomic DNA was used to prepare dot blot filters with the Minifold I dot blot apparatus (Schleicher & Schuell, Inc.). These filters were subsequently analyzed to determine approximate Lim2 copy number. They also facilitated and corroborated the phenotype, distinguishing hemizygotes from homozygotes.

Isolation of lens total RNA

Total RNA was isolated from mouse lenses using RNAzol B (Tel-Test, Inc., Friendswood, TX). Briefly, two adult lenses or 4 embryonic lenses were homogenized in 0.8 ml RNAzol B at room temperature. Chloroform (0.08 ml) was then added and the samples incubated at 4 °C for 5 min. Samples were then spun for 15 min at 4 °C in a microcentrifuge at 11,000 RPM to separate the RNA-containing aqueous phase from the DNA- and protein-containing organic phase. The aqueous phase was then carefully pipetted into a separate tube containing 0.4 ml 2-propanol and incubated overnight at 4 °C to precipitate the RNA. The RNA was then pelleted by centrifugation at 4 °C and 11,000 RPM in a microcentrifuge for 15 min. The pellet was washed once at room temperature with one ml 80% ethanol and re-pelleted as above. The final pellet was dried and resuspended in 50 μl of nuclease-free water. The purity and concentration of RNA was determined using a DU 640 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). Typical yields were 20-50 μg of total RNA per preparation.

RT-PCR analysis of lens total RNA

Approximately 500 ng of lens total RNA was used as template in RT-PCR reactions, using the Superscript One-Step RT-PCR System (Life Technologies) and 10 pmoles of each of two Lim2-specific primers. RT-PCR was carried out in a PTC-100 DNA Engine (MJ Research, Watertown, MA). The following cycling conditions were used: 20 min reverse transcription at 50 °C followed by PCR amplification consisting of an initial denaturation at 94 °C for 120 s followed by 60 cycles of 30 s at 94 °C, 30 s at 62 °C, 30 s at 70 °C followed by 5 min final extension at 72 °C. The primers used amplified an ~320 bp fragment of the Lim2 mRNA which spanned the region of the To3 mutation. As a control, each RNA sample was also amplified using primers designed to amplify an ~395 base pair fragment of mouse β-actin mRNA.

Lim2 RT-PCR products were digested with both Pst I and BstE II in order to verify identity and distinguish endogenous Lim2 product from transgenic product, respectively. β-actin RT-PCR products were digested with Bgl II to verify identity.


Generation and characterization of the Lim2To3 transgene

Using a bovine Lim2 cDNA probe, ~1 x 106 clones of a commercially available 129/SvJ murine genomic library were screened. A single clone with an ~18 kb insert was isolated and characterized by limited restriction endonuclease mapping and DNA sequencing. The insert was trimmed to ~6.3 kb, encompassing the entire murine Lim2 gene: ~400 bases of upstream promoter sequence, all coding exons and intervening introns, and 3' noncoding sequences through the polyadenylation signal. A restriction endonuclease map of the gene is shown in Figure 1.

Based upon the restriction map of the gene, a strategy was devised to introduce the To3 mutation into this Lim2 gene cassette, generating a Lim2To3 transgene. The 5' end of the transgene (encompassing the promoter, exon 1, intron A, exon 2, and a portion of intron B) was amplified from To3/To3 genomic DNA. This fragment was then joined to a 4 kb fragment (isolated from the 6.3 kb insert and including the remaining 3' end of the normal Lim2 gene). A schematic of this strategy is depicted in Figure 2. Restriction and sequence analysis of the PCR-engineered portion of the transgene cassette verified the presence of the To3 mutation in exon 2 and the absence of any other mutations.

The short stretch of 5' promoter sequence in the clone raised the concern that the transgene may not possess sufficient promoter activity and specificity. Therefore, we decided to address these concerns before proceeding. Various lengths of the murine upstream promoter were cloned into the promoterless CAT reporter vector pSVOATCAT and transfected into primary chicken lens epithelial cell cultures. As a tissue-specific control, chicken fibroblasts were also transfected with the promoter constructs. Cells were harvested and assayed for CAT and β-Gal activity 72 h after transfection. As illustrated in Figure 3A, a reasonable level of promoter activity was demonstrated to be associated with the 400 bp promoter region. This activity was only observed when transfected into lens cell cultures and not fibroblast cultures (Figure 3B), indicating at least some degree of tissue specificity. The viral promoter of SV40 was used in the fibroblast cultures as a positive control for the system. Similar results (data not shown) were obtained using N/N1003A rabbit lens epithelial cell cultures [15].

Generation and screening of Lim2To3 transgenic mice

The transgene cassette was then microinjected into fertilized single-cell (C57BL/6X SJL)F2 hybrid mouse eggs to generate potential founder mice. DNA was isolated from tail clippings of potential founder weanlings (~20 days old). These genomic DNAs were used along with Lim2-specific primers to PCR amplify an ~2.2 kb Lim2 gene fragment encompassing exon 2 and flanking intronic sequences.

A non-transgenic animal possesses two normal copies of the endogenous Lim2 gene, which can be amplified but not digested by BstE II. A transgenic animal possesses transgenic Lim2To3 copies as well as both normal copies of the endogenous Lim2. Both transgenic and endogenous Lim2 genes are therefore amplified from the genomic DNA of transgenic animals. Only the portion of product representing the transgene, however, possesses the To3 mutation and is digestible by BstE II. Restriction of the amplification products with BstE II thus allowed the presence of transgenic DNA to be determined.

Wild type parental genomic DNA produced a 2.2 kb amplification product, all of which remained intact upon BstE II restriction. Hemizygous and homozygous transgenic animals produced a product identical in size, but some of which could be restricted with BstE II to provide fragments with approximate molecular weights of 1.3 kb and 900 bases. PCR and restriction products are shown in Figure 4.

Following dilation of the iris, weanlings were examined for the presence of lenticular abnormalities, specifically opacity. Two founder mice, generated from two independent microinjections were identified. Both were positive for the presence of the LimTo3 transgene as assayed by PCR and both exhibited a similar bilateral dense nuclear cataract, with extension into the cortex as pictured in Figure 5B and Figure 5C. The remaining 20 potential founders were all lacking the transgene as determined by the PCR/BstE II restriction assay and none displayed any defects of the ocular lens.

Characterization of Lim2To3 transgenic mice

The two identified founder mice were backcrossed to C57BL/6 mice in order to test for germline transmission of the transgene and cataract. Offspring from these matings were then scored by both DNA analysis and ophthalmic exam as described above. Offspring of the first founder, 26011F, revealed transmission of both the transgene and cataract in a Mendelian fashion, approximately half of the offspring inheriting both transgene and cataract. However, the second founder, 32006F, failed to transmit the transgene and cataract to any offspring, and was thus excluded from further study.

Intercrosses between founder 26011F and hemizygous transgenic offspring produced litters demonstrating a perfect concordance between presence of the Lim2To3 transgene and cataract. Furthermore, 25% of the mutant transgenic offspring exhibited a more severe total cataract and severe microphthalmia as shown in Figure 5F.

Dot blot analysis using a bovine Lim2 cDNA probe revealed that these animals were transgenic homozygotes that received copies of the transgene from both mother and father. Dot blot analysis also provided an estimation of ~2 copies of transgene in transgenic hemizygotes. More importantly, this analysis procedure proved to be a reproducible method of distinguishing wild type, hemizygous transgenic, and homozygous transgenic animals. An example of a dot blot is shown in Figure 6.

Further analysis of greater than 100 offspring produced from intercrosses between founder and offspring as well as from intercrosses between siblings, demonstrated that the transgene and associated cataract were inherited with perfect concordance as semi-dominant Mendelian traits. One fourth of these offspring were wild type, lacking the transgene and exhibiting no lens abnormalities. One half were transgenic hemizygotes, possessing the transgene and exhibiting cataract. One fourth were transgenic homozygotes, possessing twice as much transgene and exhibiting both a more severe cataract and severe microphthalmia. The cataractous phenotype was fairly consistent: a dense nuclear cataract with extension into the cortex in hemizygotes and a total cataract in homozygotes. However, there was occasionally some variation in the hemizygous phenotype such as distinct sutural opacities and "blistering" of the lens as pictured in Figure 5D and Figure 5E.

Dissection of eyes and lenses revealed that there was a less obvious but significant reduction in the size and mass of hemizygous as well as homozygous transgenic eyes and lenses as pictured in Figure 7. Both eyes were collected from 38 total offspring and massed. The offspring comprised 2 mixed litters from 2 independent To3/+ X To3/+ crosses. Means and standard deviations for wildtype, hemizygous transgenic and homozygous transgenic litter mates are given in Table 1. Hemizygous transgenic animals exhibited a 38% reduction in mass compared to non-transgenic litter mates. Homozygous transgenic animals exhibited a 63% reduction in mass compared to non-transgenic animals. The variance (standard deviation) of the mean masses are statistically equivalent and paired t-tests reveal that the means are statistically distinct at the 95% significance level. Table 1 also exemplifies the Mendelian pattern of inheritance described above.

Histological analysis of Lim2To3 transgenic lenses

Lenses were collected from litter mates of embryonic ages E15 and E19 and from litter mates of postnatal age P18. Fixed sections were then analyzed using light microscopy. Phenotypes were corroborated by genotyping dot blot analysis of genomic DNA as described above. Representative sections from wild type, hemizygous transgenic, and homozygous transgenic mice are shown in Figure 8. At E15 (Figure 8A), there was an obvious difference in the size of the lens and eyes of the transgenic mutants, as compared to a wild type litter mate. There also appears to be a thickening, or perhaps multi-layering of the epithelium and a disorganization of the lens bow region in the transgenic mutants at this age. By E19 (Figure 8B), lenticular abnormalities are even more apparent. By P18 (Figure 8C), transgenic mutant lenses are grossly abnormal. The microphthalmic size of the mutant lenses was obvious at all stages examined.

Integration sites

Southern blot analysis was performed on genomic DNA isolated from wild type, original founder (26011F), and homozygous transgenic mice to determine the number of integration sites and corroborate an approximation of the transgene copy number. BamH I does not restrict the Lim2To3 transgene cassette nor the endogenous murine Lim2 gene. As shown in Figure 9, wild type genomic DNA digested with BamH I revealed a single fragment ~12 kb in size on a Southern blot probed with a radiolabeled Lim2 cDNA (Figure 9A, lane 1). Original founder (26011F) and homozygous transgenic genomic DNA, when treated the same, revealed 2 single fragments. One of these fragments was the same size seen in the wild type DNA and therefore represents the endogenous normal Lim2 gene. The second, larger, fragment, of at least 15-18 kb represents a concatemer of transgenic Lim2 cassettes integrated into a single chromosomal site (Figure 9A, lanes 2 and 3).

Stu I cuts once within the murine Lim2 gene, about 2 kb downstream from the beginning of exon 1. Southern analysis of wild type genomic DNAs digested with Stu I revealed 2 hybridizing fragments of about 9.5 and 4 kb (Figure 9B, lane 1). Analysis of original founder (26011F) and homozygous transgenic DNAs revealed 5 hybridizing fragments of about 4, 4.4, 6.5, 9.5, and 13 kb. Two of these were identical in size to those observed in the wild type samples, and therefore represent the endogenous Lim2 gene. The additional three fragments represent the integrated transgenic DNA (Figure 9B, lanes 2 and 3). In order to obtain three fragments there have to be two copies of the transgene, each being cut once within the transgene cassette and each flanked by a native Stu I site in the flanking DNA of the chromosomal integration site. This data therefore corroborates the dot blot Southern estimate of two copies of the transgene.

Founder 32006F revealed restriction patterns with both BamH I and Stu I that were distinct from both wild type and transgenic animals derived from the 26011F founder (not shown). This indicates that the Lim2To3 transgene is integrated in a different chromosomal site in this mouse and that the resulting cataract is due to presence of the transgene and not the site of integration.

RT-PCR assay of Lim2To3 transgene expression

As the To3 mutation results in a single amino acid substitution within the MP19 polypeptide, it was highly unlikely that the mutant protein would be distinguishable from the wild type protein using antibodies. We, therefore, assayed for transgene mRNA expression by RT-PCR of total mouse lens RNA using Lim2-specific primers. Primers were designed to amplify an approximately 320 bp fragment of the mRNA extending from the start of the coding region in exon 2 to the end of exon 3 as depicted in Figure 10. Potential contaminating genomic DNA in the RNA preps was eliminated as a concern because the two primers used in RT-PCR amplification are separated by a large intron. If any genomic DNA was present in the RNA and amplified, it would produce a much larger product that could be distinguished from the mRNA amplification product. Mouse β-actin primers were used to amplify a fragment similar in size as a positive control in the RT-PCR assay.

As the only difference between endogenous Lim2 mRNA and transgenic Lim2To3 mRNA is a single nucleotide, the amplification products once again had to be distinguished from each other by BstE II restriction analysis. As shown in Figure 11, restriction of transgenic RT-PCR amplification product yielded a fragment that was ~45 bases shorter, as expected (Figure 11, red arrowheads).

While the ratio of transgenic Lim2To3 mRNA RT-PCR product to endogenous Lim2 mRNA product was very low, it was reproducible from RNAs isolated from E18 embryonic lenses as well as weanling (~20 day old) and adult (>20 day old) lenses. The identity of both Lim2 and β-actin RT-PCR products was verified by digesting with restriction enzymes unique to each product and obtaining the expected fragment patterns.


While insertional mutagenesis has not been completely ruled out, the identification of two independent founders with distinct transgene integration sites and cataractous phenotype makes this a highly unlikely possibility. Combined, the identification of two independent transgenic founders exhibiting cataract, perfect concordance between transgene and cataract in transgenic offspring from founder 26011F, and detection of Lim2To3 mRNA via RT-PCR all support a causative relationship between the To3 mutation in the Lim2 gene and cataractogenesis and microphthalmic eye development. We, therefore, propose that To3 be designated as a mutant allele of the Lim2 gene and notated as Lim2To3.

Light microscopic analysis of lens sections indicated a gross disorganization of secondary fibers that was apparent as early as embryonic day E15. Sections from mutant lenses at embryonic days E15 and E19 and postnatal day P18 all show gross disorganization of fibers as compared to wild type sections. This disorganization of the lens is very similar to that observed in the original To3 mice [8]. Also, dissected lenses revealed that homozygous transgenic mice have a lens that is about the same size as the nucleus of a normal or hemizygous transgenic lens. These data can be interpreted in one of two ways. They could indicate that primary and initial secondary fiber formation and organization, which occur between embryonic days E12 and E13, is normal and that later secondary fiber formation and organization is perturbed. They could also indicate a subtle defect in primary and early secondary fiber formation and organization that is amplified as each new layer of secondary fibers is laid down in the developing lens. Further analysis of lens sections using higher resolution techniques such as SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy) should provide a clearer answer to this question. At the very least, however, it is safe to conclude that later secondary fiber formation and organization is grossly perturbed in the Lim2To3 transgenic lenses and is visible by light microscopy by embryonic day E15.

The fact that some phenotypic variation was observed in transgenic hemizygotes should be noted as it indicates the possibility of extragenic influences on the precise nature of the phenotype. There could be other genes that encode proteins that either interact with or modify the MP19 protein in some fashion. These genes could have allelic variants that modify the effects of mutant MP19 protein in the lens differently. Regardless, the variation is not particularly surprising as it has been documented in human hereditary cataract pedigrees as well [16].

An unexpected result in these experiments was the finding of such a low level of Lim2To3 transgenic mRNA expression as determined by RT-PCR. Based upon dot blot and classical Southern analysis of transgenic genomic DNA, there are approximately 2 copies of the Lim2To3 transgene present in the 26011F founder and its hemizygous transgenic offspring. As copy number typically has a rough correlation with expression level in transgenic mice, we expected to see similar levels of transgenic and endogenous Lim2 mRNA. There are two ways to interpret this data. The first, and most plausible, interpretation is that the transgene is expressing at a strikingly lower level than the endogenous Lim2 genes. This could be an indication that the truncated Lim2 promoter associated with the transgene is less efficient than the full-length promoter of the endogenous Lim2 genes. It could also indicate that the transgene concatemer either integrated into a transcriptionally repressed region of the chromosome or was damaged in some fashion upon integration such that not all copies are capable of expressing. A second interpretation of the low levels of transgenic mRNA is that the presence of the To3 mutation results in a less stable mRNA or an mRNA that is transcribed less efficiently compared to that of the endogenous normal Lim2 gene. This seems highly unlikely, however, as the only difference between the two mRNAs is a single nucleotide change within the protein coding exon 2.

Whatever the reason behind the lower levels of transgenic mRNA as compared with endogenous Lim2 mRNA, it points to another intriguing aspect of the To3 mutation, its potency. The original To3 mutants demonstrated that one normal Lim2 allele and one mutant allele resulted in congenital cataract. If the mRNA level is in fact representative of protein levels, an unproven assumption at this point, our transgenic animals demonstrate that only a very small percentage of the total population of MP19 molecules needs to be altered in order to have severe consequence on normal lens development.

The RT-PCR data, interpreted with the above assumption, also strengthens our previous hypothesis that the mutant allele results in a deleterious alteration of structure and/or function within the MP19 polypeptide rather than simply a loss. More specifically, the data is consistent with the notion that Lim2To3 is a dominant negative allele and MP19 must function as an oligomer rather than a monomer. One would not expect such a small number of mutant molecules to result in such a severe phenotype if MP19 acted as a monomer. However, if MP19 functions as an oligomer, then only a fraction of the monomers need be mutant in order to result in large numbers of abnormally functioning MP19 units. Previous crosslinking studies on both purified MP19 protein and native lens membranes indicated that MP19 could form oligomers as large as hexamers [17].

While no evidence of a precise role for MP19 within the ocular lens has been provided to date, our work indicates that MP19 protein with an altered structure and/or function has devastating effects on normal lens, and consequently, eye development. The observations that MP19 is the second most abundant intrinsic membrane protein within the ocular lens and its gene is highly conserved across vertebrate species indicates that MP19 is likely to fulfill a critical role in development and/or maintenance of the ocular lens. The generation and characterization of transgenic mice with a targeted ablation of the Lim2 gene should definitely answer the question of whether or not MP19 plays an essential role within the ocular lens. This work is already in progress.

There is now enough cumulative data for Lim2 to be considered a likely candidate gene for human hereditary cataract worthy of further and more intensive investigation. Also, Lim2To3 transgenic mice can now be added to the rapidly growing group of cataractous mice to serve as a model for the development and testing of therapeutic methods for recovery of lens transparency in the future. Furthermore, these mice may prove to be useful in deciphering the functional role of MP19 as well as the possible involvement or interaction of other lens proteins with MP19 during normal and/or cataractogenic development.


We would like to thank Adell Mills for his technical assistance with the preparation of lens sections for histological analyses. This research was supported in part by National Institutes of Health grants R01 EY11516, R01 EY12301, R01 EY05314, T32 EY07092, P30 EY06360, C06 EY06307, the Knights Templar Educational Foundation of Georgia, Inc., and a Departmental Grant from Research to Prevent Blindness, Inc.


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