Application of transgenic mice to the molecular pathogenesis of cataract

One of the most prevalent disorders that cause blindness worldwide is cataract,and its essence is the visual disorder caused by the opacity of the lens.The significant degree of variation in cataracts and the fact that a variety of factors can impact a patient’s lens transparency make it especially crucial to investigate the pathogenesis of cataracts at the molecular level.It has been found that more than 60 genes are linked to the formation of cataracts,and the construction of a transgenic mouse model of cataract similar to the selection of human lens clouding due to a variety of causes has become an important means of studying the pathogenesis of cataract.Therefore,the research on the application of transgenic mice to the molecular pathogenesis of cataracts will be the main topic of this review of the literature.

Antiviral treatment of hepatitis B virus-transgenic mice by a marine organism, Styela plicata

AIM： To evaluate the antiviral effect of the effective ingredient of Styela plicata in a murine model of hepatitis B virus carrier. METHODS： HBV-transgenic mice were divided into 3 groups （control group, lamivudine treatment group and the effective ingredient of Styela plicata treatment group） and assigned to receive normal diet, lamivudine or the effective ingredient of Styela plicata for consecutive weeks. Serum hepatitis B surface antigen was detected by enzyme-linked immunosorbent assay （ELISA） method. Serum HBV DNA was detected by real-time polymerase chain reaction （RT-PCR）. Serum T helper （h） 1 cytokine interleukin （IL）-2 and Th2 cytokine IL-6 were detected by the quantitative sandwich enzyme immunoassay technique. Another group of HBV-transgenic mice was assigned to receive the effective ingredient of Styela plicata for consecutive weeks. The histology of liver tissue was evaluated before and after treatment. RESULTS： Twelve weeks after starting the therapy, serum hepatitis B surface antigen was significantly lowered in Styela plicata -treated mice and lamivudine-treated mice compared with the mice receiving normal diet （F12wk = 88.81, P12wk = 0.000 〈 0.01）. Serum HBV DNA was significantly lowered in Styela plicata -treated mice and lamivudine-treated mice compared with the mice receiving normal diet （F12wk = 20.71, P12wk = 0.000 〈 0.01）. However, like lamivudine, the effective ingredient of Styela plicata could not inhibit the replication of HBV completely. A rebound phenomenon of hepatitis B surface antigen and HBV DNA in sera could be found 4 wk after withdrawal of medication. Eight weeks after starting the therapy, serum levels before and after Styela plicata treatment of IL-2 were 2.41 ± 0.38 and 10.56 ± 0.78 ng/L, respectively （t8wk = -16.51, P8wk = 0.000 〈 0.01）. Compared with the serum levels of IL-2 in the normal diet-treated mice （2.48 ± 0.17 ng/L; t8wk = 13.23, P8wk = 0.000 〈 0.01）. Serum levels before and after Styela plicata treatment of IL-6 were 63.62 ± 6.31 and 54.52 ± 6.22 ng/L, respectively, compared with the serum levels of IL-6 in the normal diet-treated mice （60.84 ± 4.21 ng/L）. Histological analysis of liver from Styela plicata-treated HBV-transgenic mice also showed catabatic status in inflammation and hepatitis B surface antigen. CONCLUSION： Styela plicata may be an effective anviral medicine in treating chronic hepatitis B.

Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABAA receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

Generation of the regulatory protein rtTA transgenic mice

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome 'immune tolerance' formed during the embryonic development and 'immune escape' against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

Tetrahydroxy Stilbene Glucoside Ameliorates Cognitive Impairments and Pathology in APP/PS1 Transgenic Mice

Cognitive impairment is the main clinical manifestation of Alzheimer's disease(AD),and amyloid-β(AB)deposition and senile plaques are the characteristic neuropathological hallmarks in AD brains.This study aimed to explore the effect and mechanism of tetrahydroxy stilbene glucoside(TSG)on cognitive function in APP/PS 1 mice during long-term administration.Here,we treated APP/PS1 model mice of AD with different doses of TSG(50 mg/kg and 100 mg/kg)for 5 to 17 months by gavage,and we further observed whether TSG could ameliorate the cognitive decline in APP/PS1 mice using behavioral tests,and investigated the possible mechanisms by immunohistochemistry and Western blotting.Our results showed that TSG treatment rescued the spatial and non-spatial learning and memory impairments of APP/PS1 mice at Morris water maze test and novel object recognition test.Furthermore,Aβ40/42 deposition in the cortex and hippocampus of APP/PS1 mice treated with TSG was significantly reduced compared to the wild type mice using the immunohistochemical technique.Finally,Western blotting showed that TSG primarily decreased the APP expression to avoid the Aβplaque deposition in the cortex and hippocampus of mice.These results reveal the beneficial effects of TSG in APP/PSI-AD mice,which may be associated with the reduction of Aβdeposits in the brain.

Establishment of Hamster-and Human-PRNP Transgenic Mice

Objective To create transgenic mice expressing hamster- and human-PRNP as a model tor understanding the physiological function and pathology of prion protein （PrP）, as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies （TSEs）. Methods Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin （H ＆ E） staining and immunohistochemical （IHC） methods. Results Integrated PRNP copy number in various mouse lines was 53 （Tg-haPrP1）, 18 （Tg-huPrP1）, 3 （Tg-huPrP2）, and 16 （Tg-huPrP5）, respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs. Conclusion We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.

Carboxymethytl Pachymaram Up-Regulates Dendritic Cell's Function in Hepatitis B Virus Transgenic Mice in vitro

The effect ofcarboxymethytl pachymaram （ CMP ） on the function of dendritic cells（DCs） derived from spleens of hepatitis B virus transgenic mice are studied in vitro. The phenotypes of DCs are tested by flow cytometry （FCM）, cytokines measured by ELISA. The expression of DCs＇ phenotypes in IdBV transgenic mice are low （CD80^＋CD11c^＋：59.12±11.53 vs 9,60±4.53, p〈0.01; CD80^＋ MHC-Ⅱ^＋： 44.86±12.31 vs 9.80±5,72, p〈0.01, normal mice vs HBV transgenic mice）, the ability of DCs stimulating T lymphocytes proliferation decreases （0.37±0.11 vs 0.20±0,11, p〈0.05, normal mice vs HBV transgenic mice）, levels of IL-12 and IFN-y decrease whereas the level of IL-10 increases; CMP can enhance DCs＇ ability of stimulating T lymphocytes proliferation, facilitate the secretion of IL-12 and IFNp, inhibit the secretion of IL-10, thus up regulates DCs function. The results show a good prospective use of CMP on the treatment of chronic hepatitis B.

Protective Effects of Overexpression of bcl-xl Gene on Local Cerebral Infarction in Transgenic Mice Undergoing Permanent Occlusion of Middle Cerebral Artery

In order to investigate the protective effects of the overexpression of bcl-xl gene on local cerebral infarction in the transgenic mice subject to permanent occlusion of middle cerebral artery, the models of bcl-xl transgenic mice were established and subjected to cerebral infarction by intraluminal occlusion of the middle cerebral artery. The infarct volume and the neurological scores were observed and comparison between the wild type mice and the transgenic mice was made. It was found that the infarct volume and the neurological scores in the transgenic mice were significantly decreased as compared with those in the wild type mice. It was suggested that the overexpression of bcl-xl gene in transgenic mice could reduce the infarct volume and improve the neurological function of the mice.

Muscle hypertrophy in transgenic mice due to over-expression of porcine myostatin mutated at its cleavage site

Myostatin, a member of the transforming growth factor beta（TGF-β） superfamily, is a dominant inhibitor that acts to limit skeletal muscle growth and development. In this study, we generated transgenic mice that express porcine myostatin containg mutations at its cleavage site（RSRR） to evaluate its effect on muscle mass. Results showed that the weight of four skeletal muscles including gastrocnemius, rectus femoris, tibialis anterior, and pectoralis increased by 17.83 and 28.39%, 21.76 and 28.70%, 34.31 and 41.62%, 53.21 and 27.54% in transgenic male and female mice, respectively, compared to their corresponding non-transgenic control mice. Measurement of muscle fiber size and number indicated that the mean myofiber size increased by 50.73 and 61.30% in transgenic male and female mice respectively compared to the non-transgenic controls. However, there was no difference in the number of myofiber between transgenic and non-transgenic male mice. These results clearly demonstrated that the increase in skeletal muscle mass in transgenic mice is caused by hypertrophy instead of hyperplasia.

Antiviral effect of Chinese medicine jiaweisinisan in hepatitis B virus transgenic mice

AIM： To study the antiviral effect of Chinese medicine jiaweisinisan （JWSNS） on hepatitis B virus （HBV） infection in transgenic mice （TGM）. METHODS： Twenty two 6-8 wk old HBV TGM in the third generation were divided into TGM control group and TGM treated group randomly. The normal control group included ten normal BC 57L/6 mice at the same age. The mice in treated group were administrated with JWSNS at the concentration of 4 g/mL and the dosage of 50 g/kg per d for 30 d, while the mice in TGM control group and normal control group were administrated with normal saline at the same dosage and the same time. Polymerase chain reaction （PCR） was used to assess the contents of HBV DNA in serum of HBV TGM before and after treatments, whereas blot hybridization was utilized to measure the contents of HBV DNA in the liver of both HBV TGM and normal BC 57L/6 mice. RESULTS： The levels of serum HBV DNA in TGM treated group were remarkably decreased after the treatment of JWSNS （7.662±0.78 vs 5.22±3.14, P〈0.05）, while there was no obvious change after administration of normal saline in TGM control group （7.125±4.26 vs 8.932 ± 5.12, P〉 0.05）. The OD values of HBV DNA in the livers of the mice in TGM treated group were significantly lower than those of TGM control group （0.274±0.096 vs 0.432 ± 0.119, P 〈 0.01）. CONCLUSION： JWSNS exerts suppressive effects on HBV DNA in the serum and liver of TGM.

Inducible overexpression of porcine homeobox A10 in the endometrium of transgenic mice

Homeobox A10 （HOXA 10） is a well-known transcription factor that plays an important role in directing endometrial differ- entiation and establishing the conditions required for implantation. Interestingly, the expression level of HOXAIO may be associated with litter size. To study the effects of the porcine HOXAIO promoter fragment on the expression of HOXAIO gene in vivo, we generated a transgenic mouse model using pronuclear microinjection, and measured the expression of HOXAIO in the endometrium. There was no difference in the expression level of HOXAIO between transgenic and wild- type mice in the absence of hormone stimulation. However, following treatment with progesterone and estradiol benzoate, the expression level of HOXAIO was significantly increased in transgenic mice compared with that of wild-type mice. Fur- thermore, the litter size of transgenic females was larger than that of wild-type females （7.02±1.73 vs. 6.48＋1.85; P=0.14）. Moreover, the difference of litter size was greater in the later parities （7.33±1.62 vs. 6.37±2.02; P=0.08） compared with the first parity （6.76±1.81 vs. 6.61v1.67; P=0.77） between transgenic and wild-type mice. Therefore, our transgenic mouse model provides exciting insights regarding the actions of HOXAIO and its hormone-inducible promoter in vivo. The present study offers valuable proof of principle to develop transgenic pigs with a hormone-inducible promoter regulating HOXAIO to alter litter size.

Protective effects on acute hypoxic-ischemic brain damage in mfat-1 transgenic mice by alleviating neuroinflammation

Acute hypoxic-ischemic brain damage(HIBD)mainly occurs in adults as a result of perioperative cardiac arrest and asphyxia.The benefits of n-3 polyunsaturated fatty acids(n-3 PUFAs)in maintaining brain growth and development are well documented.However,possible protective targets and underlying mechanisms of mfat-1 mice on HIBD require further investigation.The mfat-1 transgenic mice exhibited protective effects on HIBD,as indicated by reduced infarct range and improved neurobehavioral defects.RNA-seq analysis showed that multiple pathways and targets were involved in this process,with the anti-inflammatory pathway as the most significant.This study has shown for the first time that mfat-1 has protective effects on HIBD in mice.Activation of a G protein-coupled receptor 120(GPR120)-related anti-inflammatory pathway may be associated with perioperative and postoperative complications,thus innovating clinical intervention strategy may potentially benefit patients with HIBD.

The Establishment of Double-Transgenic Mice that Co-Express the appA and MxA Genes Mediated by Type A Spermatogonia In vivo

Type A spermatogonial stem cells are the only immortal diploid cells in the postnatal animal that undergo self-renewal through the lifetime of an animal and transmit genes to subsequent generations. In this paper, the generation and characterization of double-transgenic mice co-expressing the Escherichia coli appA gene and human MxA gene generated via the in vivo transfection of type A spermatogonial cells were reported for the ifrst time. The dicistronic expression vector pcDNA-appA-MxA（AMP） and ExGen500 transfection reagent were injected into the testicular tissue of 7-d-old male ICR mice. The mice that underwent testis-mediated gene transfer were mated with wild-type female mice, and the integration and expression of the foreign genes in the offspring were evaluated. Transgenic mice that co-expressed appA and MxA showed a gene integration rate of 8.89%（16/180）. The transgenic mice were environmentally friendly, as the amount of phosphorous remaining in the manure was reduced by as much as 11.1%by the appA gene （P〈0.05）;these animals also exhibited a strong anti-viral phenotype.

Age-related Changes in Familial Hypertrophic Cardiomyopathy Phenotype in Transgenic Mice and Humans

β-myosin heavy chain mutations are the most frequently identified basis for hypertrophic cardiomyopathy （HCM）. A transgenic mouse model （αMHC403） has been extensively used to study various mechanistic aspects of HCM. There is general skepticism whether mouse and human disease features are similar. Herein we compare morphologie and functional characteristics, and disease evolu- tion, in a transgenic mouse and a single family with a MHC mutation. Ten male αWHC403 transgenic mice （at -5 weeks, -12 weeks, and -24 weeks） and 10 HCM patients from the same family with a β-myosin heavy chain mutation were enrolled. Morphometric, conventional echocardiographic, tissue Doppler and strain analytic characteristics of transgenic mice and HCM patients were assessed. Ten male transgenic mice （αMHC403） were examined at ages -5 weeks, -12 weeks, and -24 Weeks. In the transgenic mice, aging was associated with a significant increase in septal （0.59±0.06 vs. 0.64±-0.05 vs. 0.69±0.11 mm, P〈0.01） and anterior wall thickness （0.58±0.1 vs. 0.62±0.07 vs. 0.80-1-0.16 mm, P〈0.001）, which was coincident with a significant decrease in circumferential strain （-22%=1=4% vs. -20%-4-3% vs. -19%-4-3%, P=0.03）, global longitudinal strain （-19%-4-3% vs. -17%-4-2% vs. -16%±3%, P=0.001） and E/A ratio （1.9±0.3 vs. 1.7-4-0.3 vs. 1.4-4-0.3, P=0.01）. The HCM patients were classified into 1st generation （n=6; mean age 534-6 years）, and 2nd generation （n=4; mean age 32＋8 years）. Septal thickness （2.2±0.9 vs. 1.4±0.1 cm, P〈0.05）, left atrial （LA） volume （62±16 vs. 41±5 mL, P=0.03）, E/A ratio （0.77±0.21 vs. 1.1±0.1, P=0.01）, E/e＇ ratio （25±10 vs. 12±2, P=0.03）, global left ventricular （LV） strain （-14%±3% vs. -20%±3%, P=0.01） and global LV early diastolic strain rate （0.76±0.17 s1 vs. 1.3±0.2 s-1, P=0.01） were significantly worse in the older generation. In β-myosin heavy chain muta- tions, transgenic mice and humans have similar progression in morphologic and functional abnormali- ties. The αMHC4±3 transgenic mouse model closely recapitulates human disease.

Study on Expression of KLF4 in Her2 Over-expressed Transgenic Mice

[Objective]The aim was to study the biological characteristics of Her2 over-expressed transgenic mice and to detect the changes in expression of KLF4. [Method] On the basis of establishment of Her2 over-expressed transgenic mice, the population was expanded by propagation. The genotype of the offspring was identified by PCR, the body weight-age curve was drawn, and the expression of KLF4 （FL） and KLF4α was detected by RT-PCR. [Result] In the Her2-overex-pressed breast cancer-positive mice, the expression level of KLF4α decreased slightly in the course of breast cancer, and the expression level of KLF4 （FL） was increased significantly compared with those in normal mice. However, the expression level of KLF4 gene in the breast cancer-positive mice showed a downward trend. [Conclusion] This study will fill the gaps in the study of KLF4 at the animal model level and complete the study on relationship between KLF4 and the occurrence and development of breast cancer.

The characteristics of hDPP4 transgenic mice subjected to aerosol MERS coronavirus infection via an animal nose-only exposure device

Background: Middle East respiratory syndrome coronavirus(MERS-Co V), which is not fully understood in regard to certain transmission routes and pathogenesis and lacks specific therapeutics and vaccines, poses a global threat to public health.Methods: To simulate the clinical aerosol transmission route, h DPP4 transgenic mice were infected with MERS-Co V by an animal nose-only exposure device and compared with instillation-inoculated mice. The challenged mice were observed for 14 consecutive days and necropsied on days 3, 5, 7, and 9 to analyze viral load, histopathology, viral antigen distribution, and cytokines in tissues.Results: MERS-Co V aerosol-infected mice with an incubation period of 5-7 days showed weight loss on days 7-11, obvious lung lesions on day 7, high viral loads in the lungs on days 3-9 and in the brain on days 7-9, and 60% survival. MERS-Co V instillation-inoculated mice exhibited clinical signs on day 1, obvious lung lesions on days 3-5, continuous weight loss, 0% survival by day 5, and high viral loads in the lungs and brain on days 3-5. Viral antigen and high levels of proinflammatory cytokines and chemokines were detected in the aerosol and instillation groups. Disease, lung lesion, and viral replication progressions were slower in the MERS-Co V aerosol-infected mice than in the MERS-Co V instillation-inoculated mice.Conclusion: h DPP4 transgenic mice were successfully infected with MERS-Co V aerosols via an animal nose-only exposure device, and aerosol-and instillation-infected mice simulated the clinical symptoms of moderate diffuse interstitial pneumonia. However, the transgenic mice exposed to aerosol MERS-Co V developed disease and lung pathology progressions that more closely resembled those observed in humans.

Global view of transcriptome in the brains of aged NR2B transgenic mice

NR2B subunits are involved in regulating aging, in particular, age-related learning and memory deficits. We examined 19-month-old NR2B transgenic mice and their littermate controls. First, we detected expression of the NR2B subunit gene, Grin2b, in the neocortex of transgenic mice using real-time PCR. Next, we used microarrays to examine differences in neocortical gene expression. Pathway and signal-net analyses identified multiple pathways altered in the transgenic mice, in-cluding the P53, Jak-STAT, Wnt, and Notch pathways, as well as regulation of the actin cytoskeleton and neuroactive ligand-receptor interactions. Further signal-net analysis highlighted the P53 and insulin-like growth factor pathways as key regulatory pathways. Our results provide new insight into understanding the molecular mechanisms of NR2B regulated age-related memory storage, normal organismal aging and age-related disease.

Cerebroprotective effect of Huanglian Jiedu decoction on amyloid protein precursor/presenilin-1 double transgenic mice

Huanglian Jiedu decoction （HLJDD） has been shown to improve cerebral blood flow, and reduce lipid peroxidation damage to the brain and its energy metabolism. The present study was designed to observe the cerebroprotective effect of HL.JDD on an Alzheimer＇s disease rodent model, presenilin-1/amyloid protein precursor double transgenic mice. HLJDD reduced serum interleukin-6 and interleukin-113 levels, decreased [3-amyloid precursor protein gene and senile plaque expression, resisted oxidation, and reduced free radical-induced injury, thereby improving the learning and memory of these mice. Moreover, HLJDD at 433 mg/kg per day exhibited better effects compared with that at 865 or 216 mg/kg per day, and donepezil hydrochloride at 30 mg/kg per day. Thus, these results suggest that HLJDD may have protective effects against Alzheimer＇s disease.

Can overexpression of TGF-β1 gene change the sex ratio in transgenic mice?

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene. The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAS gave an integration efficiency of 33%. TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation. Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene. The most interesting finding of the present work is the striking deviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1. The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.

Production of Transgenic Mice by Type-A Spermatogonia-Mediated Gene Transfer

Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.