[Aprospective study of detection and clinical significance of bone marrow tumor cells in small cell lung cancer].

Objective: To investigate the detection of bone marrow tumor cells in small cell lung cancer (SCLC) patients and their relationship with clinical features, treatment response and prognosis. Methods: A total of 113patients with newly diagnosed SCLC from January 2018 to October 2022 at Beijing Chest Hospital were prospectively enrolled. Before treatment, bone marrow was aspirated and separately submitted for tumor cells detection by liquid-based cytology and disseminated tumor cells (DTCs) detection by the substrction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) platform. The correlation between the detection results of the two methods with patients' clinical features and treatment response was evaluated by Chi-square. Kaplan-Meier method was applied to create survival curves and the Cox regression model was used for multivariate analysis. Results: The positive rate of bone marrow liquid-based cytology in SCLC was 15.93% (18/113). The liver and bone metastases rates were significantly higher (55.56% vs 11.58% for liver metastasis, P＜0.001; 77.78% vs 16.84% for bone metastasis, P＜0.001) and thrombocytopenia was more common (16.67% vs 2.11%, P=0.033) in patients with tumor cells detected in liquid-based cytology than those without detected tumor cells. As for SE-iFISH, DTCs were detected in 92.92% of patients (105/113), the liver and bone metastasis rates were significantly higher (37.93% vs 11.90% for liver metastasis, P=0.002; 44.83% vs 20.23 % for bone metastasis, P=0.010), and the incidence of thrombocytopenia was significantly increased (13.79% vs 1.19%, P=0.020) in patients with DTCs≥111 per 3 ml than those with DTCs＜111 per 3 ml. The positive rates of bone marrow liquid-based cytology in the disease control group and the disease progression group were 12.00% (12/100) and 46.15% (6/13), respectively, and the difference was statistically significant (P=0.002). However, the result of SE-iFISH revealed the DTCs quantities of the above two groups were 29 (8,110) and 64 (15,257) per 3 ml, and there was no statistical difference between the two groups (P=0.329). Univariate analysis depicted that the median progression-free survival (PFS) and median overall survival (OS) of liquid-based cytology positive patients were significantly shorter than those of tumor cell negative patients (6.33 months vs 9.27 months for PFS, P=0.019; 8.03 months vs 19.50 months for OS, P=0.019, P=0.033). The median PFS and median OS in patients with DTCs≥111 per 3 ml decreased significantly than those with DTCs＜111 per 3 ml (6.83 months vs 9.50 months for PFS, P=0.004; 11.2 months vs 20.60 months for OS, P=0.019). Multivariate analysis showed that disease stage (HR=2.806, 95%CI:1.499-5.251, P=0.001) and DTCs quantity detected by SE-iFISH (HR=1.841, 95%CI:1.095-3.095, P=0.021) were independent factors of PFS, while disease stage was the independent factor of OS (HR=2.538, 95%CI:1.169-5.512, P=0.019). Conclusions: Both bone marrow liquid-based cytology and SE-iFISH are clinically feasible. The positive detection of liquid-based cytology or DTCs≥111 per 3 ml was correlated with distant metastasis, and DTCs≥111 per 3 ml was an independent prognostic factor of decreased PFS in SCLC.

[Pathological diagnosis of lung cancer based on deep transfer learning].

Objective: To establish an artificial intelligence (AI)-assisted diagnostic system for lung cancer via deep transfer learning. Methods: The researchers collected 519 lung pathologic slides from 2016 to 2019, covering various lung tissues, including normal tissues, adenocarcinoma, squamous cell carcinoma and small cell carcinoma, from the Beijing Chest Hospital, the Capital Medical University. The slides were digitized by scanner, and 316 slides were used as training set and 203 as the internal test set. The researchers labeled all the training slides by pathologists and establish a semantic segmentation model based on DeepLab v3 with ResNet-50 to detect lung cancers at the pixel level. To perform transfer learning, the researchers utilized the gastric cancer detection model to initialize the deep neural network parameters. The lung cancer detection convolutional neural network was further trained by fine-tuning of the labeled data. The deep learning model was tested by 203 slides in the internal test set and 1 081 slides obtained from TCIA database, named as the external test set. Results: The model trained with transfer learning showed substantial accuracy advantage against the one trained from scratch for the internal test set [area under curve (AUC) 0.988 vs. 0.971, Kappa 0.852 vs. 0.832]. For the external test set, the transferred model achieved an AUC of 0.968 and Kappa of 0.828, indicating superior generalization ability. By studying the predictions made by the model, the researchers obtained deeper understandings of the deep learning model. Conclusions: The lung cancer histopathological diagnostic system achieves higher accuracy and superior generalization ability. With the development of histopathological AI, the transfer learning can effectively train diagnosis models and shorten the learning period, and improve the model performance.

Cost Analysis and Determinants of Living Donor Liver Transplantation in Taiwan.

BACKGROUND: Liver transplantation (LT) has become established therapy for end-stage liver disease and small-cell hepatocellular carcinoma (HCC), relying mainly on living donor LT (LDLT) in Taiwan. The cost of LDLT varies in different countries depending on the insurance system, the costs of the facility, and staff. In this study we aimed to investigate cost outcomes and determinants of LDLT in Taiwan. METHODS: From January 2014 to December 2015, 184 LDLT patients were enrolled in a study performed at the Kaohsiung Chang Gung Memorial Hospital. Patients' transplantation costs were defined as expense from immediately after surgery to discharge during hospitalization for LDLT. Antiviral therapy and hepatitis B immunoglobulin (HBIG) for prevention of hepatitis B virus (HBV) were included, but direct-acting antiviral (DAA) therapy for hepatitis C (HCV) was excluded. RESULTS: The median total, intensive care unit (ICU), and ward costs of LT were US$64,250, $43,357, and $16,138 (currency ratio 1:30), respectively. HBV significantly increased the total cost of LT, followed by postoperative reintubation and bile duct complications. CONCLUSION: The charges associated with anti-HBV viral therapy and HBIG increase the cost of LDLT. Disease severity of liver cirrhosis showed less importance in predicting cost. Postoperative complications such as reintubation or bile duct complications should be avoided to reduce the cost of LT.

Femtosecond-induced spiral micro-structured SMS fiber structure for refractive index measurement.

A single-multi-single mode (SMS) fiber structure with spiral microgroove, fabricated by femtosecond laser inscription has been proposed and successfully employed for refractive index (RI) sensing. The multimode interference in the SMS structure is effectively affected by the external perturbation due to the microgroove, which goes deep into the core of the multimode fiber (MMF). Experimental results show that this femtosecond-induced spiral micro-structured SMS (FISM-SMS) fiber structure exhibits a linear response to eternal liquid refractive index in a large RI range of 1.3373-1.4345. The maximum sensitivity of the structure can reach to 2144 nm/RIU and can be further improved by increasing the depth of the spiral micro-grooves.

[Effects of extracellular regulated protein kinases protein and impairment of blood testis barriar stucturein of mice with exposure to decabromodiphenyl ether].

Objective: To study changes in expression of claudin-11 and proteins related to mitogen-activated protein kinase (MAPK) signaling pathways, as well as the ultrastructure of the blood testis barrier (BTB), in male ICR mice exposed to decabromodiphenyl ether (BDE-209). Methods: Fifty-two mice, 4 weeks of age, weighing 15-21 g, were provided with adaptive feeding for 1 week. Mice were randomly divided into 4 groups, named control, low-dose, medium-dose and high-dose groups. The treated groups received BDE-209, by intragastric gavage, at doses, respectively, of 100, 300 and 500 mg/kg. Mice were sacrificed after 6 weeks and organs harvested on ice, weighed and stored at -80 °C. The ultrastructure of testicular tissues was examined by electron microscopy. Western blotting was used to detect proteins related to the MAPK pathway, including p38 mitogen activated protein kinase (p38), phosphorylated p38 (p-p38), extracellular regulated protein kinase 1/2 (ERK1/2) , phosphorylated ERK1/2 (p-ERK1/2) , c-jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK) and the BTB tight junction protein claudin-11. Analyze the difference between each groups. Results: At sacrifice, the body weights in each treated group were compared with those in the control group weighing (41.14 ± 0.60) g. Compared with controls, body weights were significantly different (P<0.05) in the middle dose, at (39.97 ± 0.66) g and high dose, at (39.98± 0.55) g in control group. The coefficients of the testis were significantly lower (P<0.05) in each treated group than in controls, with values of (0.37±0.0)%, (0.31±0.05)% and (0.31±0.04)% for low-dose, medium-dose and high-dose groups, respectively. The epidymus coefficient values were also significantly lower than controls (P<0.05), with values of (0.16±0.06)%, (0.11±0.05)% and (0.07±0.03)%, respectively in the same three dose groups. Electron microscopy ultrastructure showed that, compared with the control group, the testes in the middle and high dose groups had closely connected fractures, cell edema and more vacuoles. Compared with in the control group, levels of p-p38 and p-JNK in testicular tissue were significantly increased (P<0.05). In the control group and in low-, medium- and high-dose groups, the p-p38/p38 ratios were 1.35±0.13, 3.46±0.10, 5.71±0.26 and 4.79±0.21, respectively. The corresponding p-JNK/JNK ratios were 2.07±0.0, 4.77±0.18, 3.63±0.06 and 4.85±0.15. Claudin-11 levels were significantly lower (P<0.05) than control values in each dosed group. The corresponding values in control, low-dose, medium-dose and high-dose groups were 8.33±0.36, 2.06±0.27, 3.37±0.27 and 1.55±0.19, respectively. Conclusion: BDE-209 increased expression of proteins in the MAPK pathway and decreased expression of the BTB tight junction protein claudin-11 in testicular tissue. It also caused ultrastructural damage to the Sertoli cell BTB tight junctions. This suggested that BDE-209 might damage Sertoli cells BTB through effects on the MAPK pathway.

Impact of blood pressure cuff inflation rates on flow-mediated dilatation and contralateral arm response.

Flow-mediated dilatation (FMD) is widely used as an index of nitric oxide-mediated vasodilator function, yet its methodology has not been well established. Previous research indicates that a rapid inflation of a blood pressure cuff evokes systemic vasoconstriction, as it was observed even on non-occluded contralateral arm. This would potentially contribute to the variability of FMD readings and complicate the emerging evidence that non-occluded contralateral arm fingertip temperature responses during the FMD procedure may be an indicator of the presence of coronary artery disease. To test the hypotheses that rapid inflation of a blood pressure cuff could reduce FMD values and influence contralateral vasodilatory states, 33 apparently healthy adults (18 males and 15 females, 29±6 years) were studied in two randomized FMD trials. The blood flow-occluding cuff was inflated rapidly (<1 s) in one trial or slowly over 10 s in the other trial. Arterial diameter, fingertip temperature and infrared thermography were obtained throughout each session. FMD values were not different between the rapid and slow cuff inflation trials (5.9±0.6 vs 5.9±0.4%). There were no differences in reactive hyperaemia (6.4±1.6 vs 6.2±1.7 AU), shear stress (80±20 vs 77±17 dyn cm(-2)) and fingertip temperature rebound (TR; 1.8±1.2 vs 1.9±1.0 °C) between the rapid and slow inflation. Changes in finger temperature on the contralateral (non-occluded) arm were positively associated with those on the occluded arm (r=0.26 to 0.61, P<0.05). We concluded that rates of inflating a blood pressure cuff do not affect FMD and TR response, and that neurovascular-induced vasodilatation of the contralateral arm was not observed regardless of cuff inflation rates.

Aerobic power assessment by using a 10 min heart rate control running on treadmill.

AIM: This study evaluated the reliability and validity of a proposed 10 min running speed variance test (RSVHRC) in assessing aerobic power at which the intensity was controlled at 80% of age-predicted maximal heart rate (HR). METHODS: Forty-four college students (21 men and 23 women, age: 21+/-3 years, height: 166.6+/-7.9 cm, weight: 61.7+/-9.3 kg) were recruited to undergo 2 RSVHRC test trials, and a maximal exercise test at least 24 hours apart. The test consisted of a 3-min warm-up at 1.67 km/h, followed by adjusting speed up to either at 2.5 m/s or 2.78 m/s immediately depending upon onset HR after the warm-up. HR was monitored every 30 seconds and running speed was adjusted accordingly to maintain HR (+/-5bpm) for 10 minutes. RSVHRC was determined by the slope of distance/time relationship from 3rd to 10th min. RESULTS: Pair t-test showed that there was no significant difference between 1st (2.38+/-0.58 m/s) and 2nd trial (2.40+/-0.63 m/s). Intraclass correlation coefficient (ICC) score showed that RSVHRC was highly reliable (ICC=0.98, 95% CI=0.97-0.99). Coefficient of variation, standard error of measurement (SEM), and %SEM were 4.8%, 0.12 m/s, 5.02% respectively. Additionally, a Pearson product-moment correlation coefficient demonstrated 2 trials were correlated with maximal oxygen uptake (46.6+/-8.1 mL/kg/min) at r=0.74, 0.71 (P<0.05). CONCLUSION: In conclusion, 80%HRmax RSVHRC is an easy and highly reliable submaximal exercise test that provides good validity to assess aerobic power in young and healthy population, which can be applied on treadmill setting.

Metabolic syndrome in older people in Taiwan: a hospital-based study.

BACKGROUND: The aim of this study was to investigate the prevalence of metabolic syndrome in older people in Taiwan. METHODS: This was a hospital-based, cross-sectional study. We retrospectively analysed subjects receiving self-referred health examination at one medical centre in Taiwan from 2000 to 2004. In all, 696 older people without any acute illness were enrolled in this study, 352 men (50.6%) and 344 women (49.4%). The mean age was 70.2 +/- 4.6 years (age range 65-87). Metabolic syndrome was estimated according to the criteria proposed by National Cholesterol Education Program Adult Treatment Panel III in 2001 and by International Diabetes Foundation in 2005, with the cut-off values of the waist circumference greater than or equal to either 90 or 80 cm for men or women, respectively. RESULTS: Among this group, 94% of the individuals presented with at least one condition of metabolic syndrome. The prevalence of metabolic syndrome defined by National Cholesterol Education Program Adult Treatment Panel III was 44.1% and was higher in women than in men (52.6 vs 35.8%, P < 0.001). Defined by International Diabetes Foundation, the prevalence was also 44.1% and was higher in women than in men (55.5 vs 33.0%, P < 0.001). CONCLUSION: The prevalence of metabolic syndrome remains relatively high in Taiwanese older people.

Heritabilities of the metabolic syndrome and its components in the Northern Manhattan Family Study.

AIMS/HYPOTHESIS: Growing evidence suggests that the traits comprising the metabolic syndrome have a genetic basis. However, studies of genetic contributions to the syndrome are sparse. Against this background, we sought to estimate the heritability of the metabolic syndrome and its component traits. MATERIALS AND METHODS: We investigated 803 subjects from 89 Caribbean-Hispanic families who have enrolled to date in the current Northern Manhattan Family Study and for whom metabolic syndrome information was available. Metabolic syndrome was defined in accordance with the National Cholesterol Education Program Adult Treatment Panel III (NCEP/ATPIII) criteria. Variance component methods were used to estimate age and sex-adjusted heritability of the metabolic syndrome and its components. To obtain the structures underlying the metabolic syndrome, we performed principal component factor analyses using six quantitative phenotypes included in the ATPIII definition. RESULTS: The heritability for the metabolic syndrome was 24% (p=0.009), and ranged from 16 to 60% for its five components. Factor analysis yielded two independent factors (factor 1: lipids/glucose/obesity; factor 2: blood pressure). Heritability analysis revealed significant genetic effects on both factors (44% for lipids/glucose/obesity, and 20% for blood pressure). CONCLUSIONS/INTERPRETATION: In the Caribbean-Hispanic families investigated, we demonstrated moderate and significant heritabilities for the metabolic syndrome itself, as well as for individual components and independent factors of the syndrome. These results provide evidence that could support future tasks of mapping susceptibility loci for this syndrome.

Identification of T-cell epitopes in clotting factor IX and lack of tolerance in inbred mice.

Immune responses to the factor IX protein pose problems for hemophilia B patients who develop antibodies against factor IX and for potential future treatment with gene therapy. To better define the response to human factor IX, we analyzed T-cell responses to human factor IX in factor IX knockout mice on BALB/c and C57BL/6 (B6) backgrounds, both strains having CD4+ T cells that proliferate in response to human factor IX. Surprisingly, wild-type mice have similar factor IX-recognizing CD4+ T cells. We defined a dominant CD4+ epitope for each strain (CVETGVKITVVAGEH for BALB/c and LLELDEPLVLNSYVTPIC for B6) that was recognized by both factor IX knockout and wild-type mice. While human factor IX did not cross-react with the mouse homologs of these epitopes, immunization with peptides from murine factor IX stimulated proliferation in factor IX knockout mice and wild-type mice, demonstrating a failure to delete murine factor IX-specific T cells in normal mice.

Association between obesity and hyperlipidemia among children.

BACKGROUND: This study was undertaken to show the association between obesity and hyperlipidemia among the children. METHODS: In March 2000, while conducting a comprehensive health examination, we analyzed 2011 children from the first grade of primary schools in Taichung City in Taiwan. To study the association between obesity and hyperlipidemia, the t test, chi-square analysis, and multivariate logistic regression were used. RESULTS: There were 1057 boys (52.56 percent) and 954 girls (47.44 percent). The mean age was 7.27 +/- 0.46 years. The proportion of overweight was 11.07 percent in boys and 11.64 percent in girls. The proportion of obesity was 14.19 percent in boys and 12.89 percent in girls. After controlling the other covariates, the multivariate logistic regression analysis showed that overweight was associated with a low level of high density lipoprotein cholesterol. Obesity was associated with hypertriglyceridemia, a high level of low density lipoprotein cholesterol, and a low level of high density lipoprotein cholesterol. CONCLUSIONS: Our findings disclosed that the prevalence of overweight and obesity was high in childhood. Early intervention to control and prevent childhood obesity might be warranted. Obesity was associated with hyperlipidemia in children. A wide-scale survey will be suggested in the future to establish causal-effect issues between obesiyy and hyperlipidemia.

Ultraviolet radiation and reactive oxygen generation as inducers of keratinocyte apoptosis: protective role of tea polyphenols.

Ultraviolet A (UVA) radiation produces serious damage to skin, especially to dermis, but its damage to epidermis and responsible mechanisms are not fully understood. Studies were thus undertaken to investigate the effects of UVA or reactive oxygen species (ROS) on lipid peroxidation, cell cycle, and apoptosis in primary cultured rat keratinocytes and to determine the possible protective effects of tea polyphenols (TPP). UVA or ROS increased the release of plasma enzyme lactate dehydrogenase (LDH), and increased lipid peroxidation production (malondialdehyde, MDA), but decreased the activity of glutathione peroxidase (GSH-Px), indicating that UVA or ROS were cytostatic and peroxidizing to keratinocytes. TPP stabilized and protected cell membranes from ROS or UVA by inhibiting the release of LDH, lowering MDA levels, and increasing GSH-Px activity. Flow cytometry (FCM) analysis revealed that UVA or ROS decreased the proliferative index (PI); hence the cell growth was blocked in the S/G2 phase, with an increase in the percentage of apoptosis in primary keratinocytes. TPP modified the UVA or ROS-induced changes in PI and apoptosis. TPP may be useful to protect keratinocytes from UVA irradiation. In summary, these data demonstrated that UVA damage to skin keratinocytes in vitro was similar to that for ROS and that TPP protects against UVA-induced cytotoxicity by inhibiting lipid peroxidation and apoptosis.

Flow cytometric analysis of the toxicity of nitrofen in cultured keratinocytes.

Lactate dehydrogenase (LDH) release test, 3H-thymidine (3H-TdR) and 3H-leucine (3H-Leu) incorporation tests and flow cytometric analysis (FCM) of cell cycle were employed to elucidate cellular and molecular mechanism of nitrofen-induced toxicity in cultured keratinocytes. The results showed that cell morphologic damages were observed after exposure to 1.0 mmol/L and 10.0 mmol/L nitrofen. LDH release increased in a dose- and time-dependent manner. Depressions in 3H-TdR and 3H-Leu incorporation were found even at 0.01 mmol/L, and increased with the exposure dose. Cell cycle was analyzed from the DNA- histogram with propidium iodide stain. The results showed that there was no pronounced alteration in cell cycle after cells exposed to 0.01 and 0.1 mmol/L nitrofen. At dose of 1.0 mmol/L, S phase cells increased 2 times of that of control. With the increase of dose, G2/M phase cells became to increase about 5 times of that of the control. At 1.0 mmol/L, time course of cell cycle after exposure was observed. At the beginning of exposure, cells in S phase and G2/M phase were about 8.7% and 11%. Following 24 h incubation with nitrofen, cells in S phase increased to 18.0% with almost no change in G2/M. 72 h after exposure, G2/M phase cells increased to 63.3%. The above results demonstrated that S phase and G2/M phase blockage in cultured keratinocytes after exposed to nitrofen seems of importance in the mechanism of nitrofen-induced toxicity.

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Preliminary validation of tumor cell attachment inhibition assay for developmental toxicants with mouse S180 cells.

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180 cells attachment to Concanavalin A-coated surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration that reduced attachment by 50%), of these 2 chemicals was 1.2 x 10(-3) mol/L and 1.0 mol/L, respectively. Another developmental toxicant, hydrocortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally similar to those obtained with ascitic mouse ovarian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not limit attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an alternative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

Human factor IX corrects the bleeding diathesis of mice with hemophilia B.

Mice with hemophilia B have been engineered using gene targeting techniques. These animals exhibit severe factor IX deficiency and a clinical phenotype that mirrors the human disease. We have bred the founder animals onto two different strains of mice, C57B1/6 and CD-1, and have sought to determine whether adenoviral vectors expressing human factor IX could correct the bleeding diathesis of mice with hemophilia B. Initial experiments showed that purified plasma-derived human factor IX added to murine factor IX-deficient plasma resulted in complete correction of the activated partial thromboplastin time (aPTT), and that injection of 10(11) particles of an adenoviral vector expressing human factor IX resulted in normalization of a modified aPTT in mouse plasma. As an additional method of assessing the function of human factor IX in the murine coagulation system, bleeding times were performed in normal, hemophilic, and adenoviral-treated hemophilic mice. By two different bleeding-time techniques, the treated hemophilic mice gave values identical to normal littermate controls, whereas the untreated hemophilic mice exhibited heavy blood loss and prolonged bleeding. There was a marked difference in antibody formation in the two strains of mice; 100% of the hemophilic CD-1 mice formed antibodies to human factor IX, but none of the C57B1/6 mice did. These data suggest that the C57B1/6 hemophilic mice will be more useful for gene transfer studies, while the CD-1 hemophilic mice may be of greater utility in studying the development of inhibitors.

A coagulation factor IX-deficient mouse model for human hemophilia B.

Coagulation factor IX deficiency causes hemophilia B in humans. We have used gene targeting to develop a coagulation factor IX-deficient (factor IX-knockout) mouse strain. Mouse embryonic stem (ES) cells were targeted by a socket-containing vector that replaces the promoter through exon 3 of the factor IX gene by neoDeltaHPRT, which is a functional neo gene plus a partially deleted hypoxanthine phosphoribosyl transferase minigene. Chimeric mice generated using these socket-containing ES cells transmitted the targeted factor IX gene to their female offspring. Male offspring from these females were characterized and shown to exhibit a phenotype similar to hemophilia B. This factor IX-deficient mouse strain will be useful for studying gene therapy methods and structure-function relationships of recombinant factor IX proteins in vivo.